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Effects of either PI3K or MEK inhibition as well as the antioxidant Trolox on the up-regulation of CAV1 induced by Methotrexate and <t>Etoposide</t> Colon cancer (A) HT29(US) and (B) DLD-1 cells were treated with either the PI3K inhibitor LY294002 (LY, 10 μM), the MEK inhibitor PD98059 (PD, 50 μM) or the vitamin E analog, Trolox (TR, 2 mM) for 30 min before treatment with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Cells were harvested and total protein extracts were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with antibodies against CAV1 and β-actin. The graphs show the expression of CAV1 normalized to β-actin (mean ± SEM) averaged from 3 independent experiments. Significant differences in comparison with the untreated condition (Basal) are indicated *** p ≤ 0.001, ** p ≤ 0.05, * p ≤ 0.01.
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Images

1) Product Images from "Anti-neoplastic drugs increase caveolin-1-dependent migration, invasion and metastasis of cancer cells"

Article Title: Anti-neoplastic drugs increase caveolin-1-dependent migration, invasion and metastasis of cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.22955

Effects of either PI3K or MEK inhibition as well as the antioxidant Trolox on the up-regulation of CAV1 induced by Methotrexate and Etoposide Colon cancer (A) HT29(US) and (B) DLD-1 cells were treated with either the PI3K inhibitor LY294002 (LY, 10 μM), the MEK inhibitor PD98059 (PD, 50 μM) or the vitamin E analog, Trolox (TR, 2 mM) for 30 min before treatment with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Cells were harvested and total protein extracts were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with antibodies against CAV1 and β-actin. The graphs show the expression of CAV1 normalized to β-actin (mean ± SEM) averaged from 3 independent experiments. Significant differences in comparison with the untreated condition (Basal) are indicated *** p ≤ 0.001, ** p ≤ 0.05, * p ≤ 0.01.
Figure Legend Snippet: Effects of either PI3K or MEK inhibition as well as the antioxidant Trolox on the up-regulation of CAV1 induced by Methotrexate and Etoposide Colon cancer (A) HT29(US) and (B) DLD-1 cells were treated with either the PI3K inhibitor LY294002 (LY, 10 μM), the MEK inhibitor PD98059 (PD, 50 μM) or the vitamin E analog, Trolox (TR, 2 mM) for 30 min before treatment with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Cells were harvested and total protein extracts were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with antibodies against CAV1 and β-actin. The graphs show the expression of CAV1 normalized to β-actin (mean ± SEM) averaged from 3 independent experiments. Significant differences in comparison with the untreated condition (Basal) are indicated *** p ≤ 0.001, ** p ≤ 0.05, * p ≤ 0.01.

Techniques Used: Inhibition, SDS Page, Western Blot, Expressing

Effect of MEK and Src family kinase inhibition, as well as the anti-oxidants Trolox and Tiron on cell migration induced by Methotrexate and Etoposide (A) HT29(US) or (B) DLD-1 cells (6 x 10 5 ) were seeded in 6 cm plates 24 h before treatment with the MEK inhibitor PD98059 (PD, 50 μM), the Src family kinase inhibitor PP2 (1 mM), the vitamin E analog Trolox (TR, 2 mM) or the superoxide scavenger Tiron (Ti, 4 mM) for 30 min (PD, TR and Ti) or 1 h (PP2) before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells (2 x 10 5 ) were then seeded in Boyden chambers coated with fibronectin (2 μg/ml) and allowed to migrate for 7 h (HT29(US) cells) or 5 h (DLD1 cells). The cells that migrated through the pores were stained and counted. Values were normalized to those obtained for cells without treatment (Basal). The graphs show the averages of results from 3 independent experiments (mean ± SEM). Significant differences are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Effect of MEK and Src family kinase inhibition, as well as the anti-oxidants Trolox and Tiron on cell migration induced by Methotrexate and Etoposide (A) HT29(US) or (B) DLD-1 cells (6 x 10 5 ) were seeded in 6 cm plates 24 h before treatment with the MEK inhibitor PD98059 (PD, 50 μM), the Src family kinase inhibitor PP2 (1 mM), the vitamin E analog Trolox (TR, 2 mM) or the superoxide scavenger Tiron (Ti, 4 mM) for 30 min (PD, TR and Ti) or 1 h (PP2) before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells (2 x 10 5 ) were then seeded in Boyden chambers coated with fibronectin (2 μg/ml) and allowed to migrate for 7 h (HT29(US) cells) or 5 h (DLD1 cells). The cells that migrated through the pores were stained and counted. Values were normalized to those obtained for cells without treatment (Basal). The graphs show the averages of results from 3 independent experiments (mean ± SEM). Significant differences are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Inhibition, Migration, Staining

Ex vivo treatment of colon carcinoma cells with Methotrexate and Etoposide increases the number of CAV1-expressing cells in ascites fluid and metastasis in vivo (A) Schematic summarizing events in the intraperitoneal carcinomatosis assay: Seven week-old BalbC/NoD/SciD mice (5 mice per group) were injected intraperitoneally with 1 x 10 6 sh-Scramble or sh-CAV1 (#5) HT29(US) cells treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h prior to injection. After 21 days, the animals were euthanized and paracentesis was analyzed. (B) The graph shows the number of viable cells in the ascitic fluid in each condition. (C) Representative images at 21 days post-cell injection showing the intestine, pancreas, spleen and stomach of mice injected with HT29(US) sh-Scramble (upper panel) or HT29(US) sh-CAV1 (#5) (lower panel) that prior to injection were either not treated (Basal) or treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. (D) The graph shows the total mass of the solid tumors and the infiltrated organs (intestine, pancreas, spleen and stomach) of mice injected with HT29(US) sh-Scramble or HT29(US) sh-CAV1 (#5) that were previously either not treated (Basal) or treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h, as well as the total weight of the organs in non-treated mice (NT). Significant differences are indicated, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Ex vivo treatment of colon carcinoma cells with Methotrexate and Etoposide increases the number of CAV1-expressing cells in ascites fluid and metastasis in vivo (A) Schematic summarizing events in the intraperitoneal carcinomatosis assay: Seven week-old BalbC/NoD/SciD mice (5 mice per group) were injected intraperitoneally with 1 x 10 6 sh-Scramble or sh-CAV1 (#5) HT29(US) cells treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h prior to injection. After 21 days, the animals were euthanized and paracentesis was analyzed. (B) The graph shows the number of viable cells in the ascitic fluid in each condition. (C) Representative images at 21 days post-cell injection showing the intestine, pancreas, spleen and stomach of mice injected with HT29(US) sh-Scramble (upper panel) or HT29(US) sh-CAV1 (#5) (lower panel) that prior to injection were either not treated (Basal) or treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. (D) The graph shows the total mass of the solid tumors and the infiltrated organs (intestine, pancreas, spleen and stomach) of mice injected with HT29(US) sh-Scramble or HT29(US) sh-CAV1 (#5) that were previously either not treated (Basal) or treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h, as well as the total weight of the organs in non-treated mice (NT). Significant differences are indicated, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Ex Vivo, Expressing, In Vivo, Mouse Assay, Injection

CAV1 silencing precludes the increase in cell migration induced by Methotrexate and Etoposide in colon cancer cell lines Parental, sh-Scramble and sh-CAV1 (#5) (A) HT29(US) or (B) DLD-1 cells (6 x 10 5 ) were seeded in 6 cm plates 24 h before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells (2 x 10 5 ) were then seeded in Boyden chambers coated with fibronectin (2 μg/ml) on the lower side and allowed to migrate for 7 h (HT29(US) cells) or 5 h (DLD1 cells). The cells that migrated through the pores were stained and counted. Values obtained were normalized to those obtained for parental cells without treatment. The graphs show the averages of values from 3 independent experiments (mean ± SEM). Significant differences are indicated ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: CAV1 silencing precludes the increase in cell migration induced by Methotrexate and Etoposide in colon cancer cell lines Parental, sh-Scramble and sh-CAV1 (#5) (A) HT29(US) or (B) DLD-1 cells (6 x 10 5 ) were seeded in 6 cm plates 24 h before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells (2 x 10 5 ) were then seeded in Boyden chambers coated with fibronectin (2 μg/ml) on the lower side and allowed to migrate for 7 h (HT29(US) cells) or 5 h (DLD1 cells). The cells that migrated through the pores were stained and counted. Values obtained were normalized to those obtained for parental cells without treatment. The graphs show the averages of values from 3 independent experiments (mean ± SEM). Significant differences are indicated ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Migration, Staining

CAV1 silencing decreases metalloproteinase activity induced by Methotrexate and Etoposide in colon cancer cell lines sh-Scramble and sh-CAV1 (#5) (A) HT29(US) or (B) DLD1 cells (6 X 10 5 ) were seeded in 6 cm plates 24 h before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells were harvested and total protein extracts were subjected to gelatin zymography. The graphs show the densitometric analysis of gelatinolytic activity detected at 92 kDa (pro-MMP9) and 72 kDa (pro-MMP2) averaged from 3 independent experiments (mean ± SEM). Significant differences are indicated, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: CAV1 silencing decreases metalloproteinase activity induced by Methotrexate and Etoposide in colon cancer cell lines sh-Scramble and sh-CAV1 (#5) (A) HT29(US) or (B) DLD1 cells (6 X 10 5 ) were seeded in 6 cm plates 24 h before treatment with 100 nM Methotrexate or 10 μM Etoposide for 48 h. Cells were harvested and total protein extracts were subjected to gelatin zymography. The graphs show the densitometric analysis of gelatinolytic activity detected at 92 kDa (pro-MMP9) and 72 kDa (pro-MMP2) averaged from 3 independent experiments (mean ± SEM). Significant differences are indicated, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Activity Assay, Zymography

Working model summarizing the main findings described in this study identifying the mechanisms by which cytotoxic drugs induce CAV1 expression Initially, CAV1 expression is repressed by methylation in the gene promoter region in tumor cells. Exposure to the anti-neoplastic drugs Methotrexate or Etoposide induces promoter demethylation that increases transcription and expression of CAV1 that is is mediated by ERK activation and ROS production. Likewise, ROS are shown to promote Src family kinase-dependent CAV1 phosphorylation that may lead to Rac1 and metalloproteinase activation, as well as increased migration, invasion and metastasis.
Figure Legend Snippet: Working model summarizing the main findings described in this study identifying the mechanisms by which cytotoxic drugs induce CAV1 expression Initially, CAV1 expression is repressed by methylation in the gene promoter region in tumor cells. Exposure to the anti-neoplastic drugs Methotrexate or Etoposide induces promoter demethylation that increases transcription and expression of CAV1 that is is mediated by ERK activation and ROS production. Likewise, ROS are shown to promote Src family kinase-dependent CAV1 phosphorylation that may lead to Rac1 and metalloproteinase activation, as well as increased migration, invasion and metastasis.

Techniques Used: Expressing, Methylation, Activation Assay, Migration

Anti-neoplastic drugs increase CAV1 expression in colon and breast cancer cell lines Colon cancer cells (A) HT29(US), (B) DLD-1, (C) HT29(ATCC) and breast cancer cells (D) MCF-7 were treated with 100 nM Methotrexate (MT), 10 μM Etoposide (ET), 1 μM Doxorubicin (DX), 5 nM Staurosporine (ST), 5 nM Taxol (TX) or 100 nM Cisplatin (CP) for 24 h. Cells were harvested and total protein extracts were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with antibodies against caveolin-1 (CAV1) and β-actin. The graphs show the expression of CAV1 normalized to β-actin (mean ± SEM) of 3 independent experiments. Significant differences in comparison with the untreated condition (Basal) are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 .
Figure Legend Snippet: Anti-neoplastic drugs increase CAV1 expression in colon and breast cancer cell lines Colon cancer cells (A) HT29(US), (B) DLD-1, (C) HT29(ATCC) and breast cancer cells (D) MCF-7 were treated with 100 nM Methotrexate (MT), 10 μM Etoposide (ET), 1 μM Doxorubicin (DX), 5 nM Staurosporine (ST), 5 nM Taxol (TX) or 100 nM Cisplatin (CP) for 24 h. Cells were harvested and total protein extracts were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with antibodies against caveolin-1 (CAV1) and β-actin. The graphs show the expression of CAV1 normalized to β-actin (mean ± SEM) of 3 independent experiments. Significant differences in comparison with the untreated condition (Basal) are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 .

Techniques Used: Expressing, SDS Page, Western Blot

Methotrexate and Etoposide induce an increase in CAV1 mRNA levels in colon cancer cell lines Colon cancer cells (A) HT29(US) and (B) DLD-1 were treated with 100 nM Methotrexate or 10 μM Etoposide for 12 and 24 h. CAV1 mRNA levels were evaluated by quantitative RT-PCR analysis, using β-actin as an internal control. Values obtained by analysis of three independent experiments are shown for CAV1 mRNA following standardization to β-actin (mean ± SEM) and after normalizing to the values obtained for untreated (0 h) samples. Statistically significant differences compared with the controls (time 0) are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Methotrexate and Etoposide induce an increase in CAV1 mRNA levels in colon cancer cell lines Colon cancer cells (A) HT29(US) and (B) DLD-1 were treated with 100 nM Methotrexate or 10 μM Etoposide for 12 and 24 h. CAV1 mRNA levels were evaluated by quantitative RT-PCR analysis, using β-actin as an internal control. Values obtained by analysis of three independent experiments are shown for CAV1 mRNA following standardization to β-actin (mean ± SEM) and after normalizing to the values obtained for untreated (0 h) samples. Statistically significant differences compared with the controls (time 0) are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Quantitative RT-PCR

Methotrexate and Etoposide induce CAV1 promoter demethylation in colon cancer cells (A) HT29(US) and (B) DLD-1 colon cancer cells were treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Genomic DNA (gDNA) was denatured for 10 min at 95°C and then immunoprecipitated using the anti-5-methylcytidine antibody (5mC). Affinity purified DNA was then evaluated using qPCR analysis, defining the enrichment levels as a percentage of the input material. Specific primers were used to analyze the CAV1 proximal promoter (CAV1 promoter) or negative (CSa region) and positive control regions (IAP region). Statistically significant differences compared with the control (Basal, black bars) are indicated *** p ≤ 0.001, ** p ≤ 0.01.
Figure Legend Snippet: Methotrexate and Etoposide induce CAV1 promoter demethylation in colon cancer cells (A) HT29(US) and (B) DLD-1 colon cancer cells were treated with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Genomic DNA (gDNA) was denatured for 10 min at 95°C and then immunoprecipitated using the anti-5-methylcytidine antibody (5mC). Affinity purified DNA was then evaluated using qPCR analysis, defining the enrichment levels as a percentage of the input material. Specific primers were used to analyze the CAV1 proximal promoter (CAV1 promoter) or negative (CSa region) and positive control regions (IAP region). Statistically significant differences compared with the control (Basal, black bars) are indicated *** p ≤ 0.001, ** p ≤ 0.01.

Techniques Used: Immunoprecipitation, Affinity Purification, Real-time Polymerase Chain Reaction, Positive Control

MEK inhibition reduces ROS production induced by Methotrexate and Etoposide (A) HT29(US) or (B) DLD-1 colon cancer cells (3 x 10 6 ) were seeded in 24-well plates and, after 24 h, were treated with the MEK inhibitor PD98059 (PD, 50 μM) for 30 min, added prior to treatment with 100 nM Methotrexate for 20 h or 10 μM Etoposide for the indicated time periods. Cells were washed 3 times with PBS and subsequently incubated with trypsin for 5 min. Once in suspension, cells were loaded with the probe DHR123 (1.4 μg/ml) in RPMI media without serum for 30 min and then the reaction was stopped on ice. The extent of DHR123 oxidation was determined by flow cytometry. The graphs show DHR123 fluorescence normalized to the untreated condition (Basal) (mean ± SEM) averaged from 3 independent experiments. Significant differences are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: MEK inhibition reduces ROS production induced by Methotrexate and Etoposide (A) HT29(US) or (B) DLD-1 colon cancer cells (3 x 10 6 ) were seeded in 24-well plates and, after 24 h, were treated with the MEK inhibitor PD98059 (PD, 50 μM) for 30 min, added prior to treatment with 100 nM Methotrexate for 20 h or 10 μM Etoposide for the indicated time periods. Cells were washed 3 times with PBS and subsequently incubated with trypsin for 5 min. Once in suspension, cells were loaded with the probe DHR123 (1.4 μg/ml) in RPMI media without serum for 30 min and then the reaction was stopped on ice. The extent of DHR123 oxidation was determined by flow cytometry. The graphs show DHR123 fluorescence normalized to the untreated condition (Basal) (mean ± SEM) averaged from 3 independent experiments. Significant differences are indicated *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Inhibition, Incubation, Flow Cytometry, Cytometry, Fluorescence

Methotrexate and Etoposide increase cancer cell invasion in a Src family kinase and Rac1 dependent manner (A) HT29(US) or (B) DLD1 (6 X 10 5 ) cells were seeded in 6 cm plates 24 h before pre-treatment with either the Src family kinase inhibitor, PP2 (1 mM) or with the Tiam1 inhibitor, NS (NSC 23766, 100 mM) for 60 min followed by the treatment with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. (C) HT29(US) or (D) DLD1 (6 X 10 5 ) cells were transfected with GFP (white bars) or with the Rac1 dominant-negative, RacS17N (black bars), 24 h before the treatment with 100 mM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Then, cells (2 x 10 5 ) were seeded in Matrigel-coated chambers and allowed to invade the matrix for 24 h. The cells that accumulated on the lower surface of the membrane were then stained and counted. Values obtained were normalized to those obtained for cells without treatment (Basal). The graphs show the averages of results from 3 independent experiments (mean ± SEM). Significant differences are indicated, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Methotrexate and Etoposide increase cancer cell invasion in a Src family kinase and Rac1 dependent manner (A) HT29(US) or (B) DLD1 (6 X 10 5 ) cells were seeded in 6 cm plates 24 h before pre-treatment with either the Src family kinase inhibitor, PP2 (1 mM) or with the Tiam1 inhibitor, NS (NSC 23766, 100 mM) for 60 min followed by the treatment with 100 nM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. (C) HT29(US) or (D) DLD1 (6 X 10 5 ) cells were transfected with GFP (white bars) or with the Rac1 dominant-negative, RacS17N (black bars), 24 h before the treatment with 100 mM Methotrexate (MT) or 10 μM Etoposide (ET) for 48 h. Then, cells (2 x 10 5 ) were seeded in Matrigel-coated chambers and allowed to invade the matrix for 24 h. The cells that accumulated on the lower surface of the membrane were then stained and counted. Values obtained were normalized to those obtained for cells without treatment (Basal). The graphs show the averages of results from 3 independent experiments (mean ± SEM). Significant differences are indicated, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Transfection, Dominant Negative Mutation, Staining

2) Product Images from "IKK? and IKK? Regulation of DNA Damage-Induced Cleavage of Huntingtin"

Article Title: IKK? and IKK? Regulation of DNA Damage-Induced Cleavage of Huntingtin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005768

Inhibition of IKKβ prevents proteolysis of Htt induced by etoposide. (A) MESC2.10 neurons were transduced with a control or lentivirus expressing specific anti-IKKβ shRNA. The level of IKKβ protein was examined by Western blotting with an anti-IKKβ antibody. Bottom panel shows staining of same blot for α-tubulin. (B) Inhibition of etoposide-induced Htt cleavage by silencing of IKKβ. Control or MESC2.10 neurons with silenced IKKβ were treated with etoposide for 6 hrs and examined for Htt cleavage (lanes 1 and 2) as described in Fig. 3A . Arrowhead indicates position of full length Htt, and the arrow shows the position of the major cleaved product. The second panel shows IKKγ levels used as a loading control. (C) DNA binding activity of P65 NF-κB is increased by etoposide and is suppressed by IKKα. Binding to consensus NF-κB oligonucleotides and detection was described in M M. Lanes 1–4 show p65 binding from nuclear extracts of control and lanes 5–8 are from neurons transduced with a lentivirus expressing IKKα ( Fig. 3C ). Bars indicate S.E.M. and asterisk shows significant difference between control and IKKα+ neurons treated with etoposide for 4 hr, P
Figure Legend Snippet: Inhibition of IKKβ prevents proteolysis of Htt induced by etoposide. (A) MESC2.10 neurons were transduced with a control or lentivirus expressing specific anti-IKKβ shRNA. The level of IKKβ protein was examined by Western blotting with an anti-IKKβ antibody. Bottom panel shows staining of same blot for α-tubulin. (B) Inhibition of etoposide-induced Htt cleavage by silencing of IKKβ. Control or MESC2.10 neurons with silenced IKKβ were treated with etoposide for 6 hrs and examined for Htt cleavage (lanes 1 and 2) as described in Fig. 3A . Arrowhead indicates position of full length Htt, and the arrow shows the position of the major cleaved product. The second panel shows IKKγ levels used as a loading control. (C) DNA binding activity of P65 NF-κB is increased by etoposide and is suppressed by IKKα. Binding to consensus NF-κB oligonucleotides and detection was described in M M. Lanes 1–4 show p65 binding from nuclear extracts of control and lanes 5–8 are from neurons transduced with a lentivirus expressing IKKα ( Fig. 3C ). Bars indicate S.E.M. and asterisk shows significant difference between control and IKKα+ neurons treated with etoposide for 4 hr, P

Techniques Used: Inhibition, Transduction, Expressing, shRNA, Western Blot, Staining, Binding Assay, Activity Assay

IKKs influence etoposide-induced activation of caspases. (A) Activation of caspase-3 and caspase-6. MESC2.10 neurons were treated with etoposide as in figure 3A and examined for the levels of procaspase-3 (top panel) or procaspses-6 (middle panel) by Western blotting. Arrow shows the cleaved products of procaspase-3. (B and C) Caspase-3 (B) and caspase-6 (C) activities are shown in MESC2.10 neuronal lysates. For the specific inhibitors neurons were first pretreated with 20 µM of Ac-DEVD-CHO, caspase-3 inhibitor (C3I) or 20 µM of Ac-VEID-CHO, caspase-6 inhibitor (C6I), or 5 mg/ml of sodium salicylate (NaSal) one hr prior to etoposide treatment for 6 hrs. Extracts were incubated with either caspase-3 substrate (DEVD conjugated to p-nitroanaline) or caspase-6 substrate (VEID conjugated to p-nitroanline) in a 96 well plate at 37°C for 1 hr. Enzyme activities for caspase-3 (B) or caspase-6 (C) were measured in a microplate reader. Results are shown as relative enzyme activity and represent averages of three experiments. Bars indicate S.E.M. and asterisk shows significant difference from etoposide treated control neurons (column 2), p
Figure Legend Snippet: IKKs influence etoposide-induced activation of caspases. (A) Activation of caspase-3 and caspase-6. MESC2.10 neurons were treated with etoposide as in figure 3A and examined for the levels of procaspase-3 (top panel) or procaspses-6 (middle panel) by Western blotting. Arrow shows the cleaved products of procaspase-3. (B and C) Caspase-3 (B) and caspase-6 (C) activities are shown in MESC2.10 neuronal lysates. For the specific inhibitors neurons were first pretreated with 20 µM of Ac-DEVD-CHO, caspase-3 inhibitor (C3I) or 20 µM of Ac-VEID-CHO, caspase-6 inhibitor (C6I), or 5 mg/ml of sodium salicylate (NaSal) one hr prior to etoposide treatment for 6 hrs. Extracts were incubated with either caspase-3 substrate (DEVD conjugated to p-nitroanaline) or caspase-6 substrate (VEID conjugated to p-nitroanline) in a 96 well plate at 37°C for 1 hr. Enzyme activities for caspase-3 (B) or caspase-6 (C) were measured in a microplate reader. Results are shown as relative enzyme activity and represent averages of three experiments. Bars indicate S.E.M. and asterisk shows significant difference from etoposide treated control neurons (column 2), p

Techniques Used: Activation Assay, Western Blot, Incubation, Activity Assay

A schematic diagram showing a potential signaling pathway for IKKβ-mediated Htt proteolysis in MESC2.10 neurons. (A) DNA damage activates IKKβ, which can phosphorylate Bcl-xL and enhance its degradation (arrow 1). Reduction of Bcl-xL levels triggers the activation of caspases, which cleaves Htt. IKKβ inhibition block degradation of Bcl-xL, caspases activation, and proteolysis of Htt. Similar to the inhibition of IKKβ, elevation of Bcl-xL also prevents caspase activation and Htt proteolysis. On the other hand, etoposide treatment reduces the activity of IKKα (arrow 2). This may enhance IKKβ activation and/or block expression of neuroprotective proteins that are essential for interfering with caspase activation and maintaining Htt levels. Elevated IKKα expressed from a lentivirus overcomes these deficiencies and prevents Htt proteolysis.
Figure Legend Snippet: A schematic diagram showing a potential signaling pathway for IKKβ-mediated Htt proteolysis in MESC2.10 neurons. (A) DNA damage activates IKKβ, which can phosphorylate Bcl-xL and enhance its degradation (arrow 1). Reduction of Bcl-xL levels triggers the activation of caspases, which cleaves Htt. IKKβ inhibition block degradation of Bcl-xL, caspases activation, and proteolysis of Htt. Similar to the inhibition of IKKβ, elevation of Bcl-xL also prevents caspase activation and Htt proteolysis. On the other hand, etoposide treatment reduces the activity of IKKα (arrow 2). This may enhance IKKβ activation and/or block expression of neuroprotective proteins that are essential for interfering with caspase activation and maintaining Htt levels. Elevated IKKα expressed from a lentivirus overcomes these deficiencies and prevents Htt proteolysis.

Techniques Used: Activation Assay, Inhibition, Blocking Assay, Activity Assay, Expressing

Etoposide promotes Htt proteolysis and that is inhibited by elevated IKKα expression. (A) EGFP and IKKα-transduced neurons were treated with 10 µM of etoposide for the indicated times. Extracts were examined for Htt by Western blotting. The top panel shows staining with anti-Htt (mAb 2166) antibody. The asterisk indicates full-length endogenous Htt and the arrow shows the cleaved products. The second panel shows staining for tubulin. Fold changes for full-length Htt levels were obtained by measuring the band intensity in each lane, normalized to tubulin and compared to non-treated control. (B) p53 accumulates in the nucleus of etoposide treated neurons. Nuclear extracts from MESC2.10 neurons were examined for the presence p53 by Western blotting. Lanes 1–4 are nuclear extracts from MESC2.10 neurons with EGFP and lanes 5–8 are from neurons that were transduced with IKKα lentiviruses ( Fig. 1C ). Staining with anti-lamin B1 was used to ensure equal loading (bottom panel). (C) Western blot analysis of IKKα levels in the control and IKKα-expressing neurons. MESC2.10 neuroblasts were transduced with an IKKα recombinant lentivirus and differentiated as described in M M. EGFP lentivirus was used as a control. Top panel shows the Western blot for IKKα and bottom panel is staining of the same blot for tubulin.
Figure Legend Snippet: Etoposide promotes Htt proteolysis and that is inhibited by elevated IKKα expression. (A) EGFP and IKKα-transduced neurons were treated with 10 µM of etoposide for the indicated times. Extracts were examined for Htt by Western blotting. The top panel shows staining with anti-Htt (mAb 2166) antibody. The asterisk indicates full-length endogenous Htt and the arrow shows the cleaved products. The second panel shows staining for tubulin. Fold changes for full-length Htt levels were obtained by measuring the band intensity in each lane, normalized to tubulin and compared to non-treated control. (B) p53 accumulates in the nucleus of etoposide treated neurons. Nuclear extracts from MESC2.10 neurons were examined for the presence p53 by Western blotting. Lanes 1–4 are nuclear extracts from MESC2.10 neurons with EGFP and lanes 5–8 are from neurons that were transduced with IKKα lentiviruses ( Fig. 1C ). Staining with anti-lamin B1 was used to ensure equal loading (bottom panel). (C) Western blot analysis of IKKα levels in the control and IKKα-expressing neurons. MESC2.10 neuroblasts were transduced with an IKKα recombinant lentivirus and differentiated as described in M M. EGFP lentivirus was used as a control. Top panel shows the Western blot for IKKα and bottom panel is staining of the same blot for tubulin.

Techniques Used: Expressing, Western Blot, Staining, Transduction, Recombinant

Etoposide induces proteolysis of endogenous mutant Htt in neurons striatal (Hdh Q111/Q111 ) neurons. The caspase-3 inhibitor (C3I, 20 µM) and sodium salicylate (NaSal, 5 mg/ml) were added 1 hr prior to the addition of 10 µM etoposide for 6 hrs. Processing of samples was as described in figure 3A . Top panel shows Western blot analysis of lysates for Htt. Arrow indicates the full-length Htt and the arrowhead shows the cleaved products (∼90 kDa). The second panel shows the level of Bcl-xL. IKKγ was used as loading control.
Figure Legend Snippet: Etoposide induces proteolysis of endogenous mutant Htt in neurons striatal (Hdh Q111/Q111 ) neurons. The caspase-3 inhibitor (C3I, 20 µM) and sodium salicylate (NaSal, 5 mg/ml) were added 1 hr prior to the addition of 10 µM etoposide for 6 hrs. Processing of samples was as described in figure 3A . Top panel shows Western blot analysis of lysates for Htt. Arrow indicates the full-length Htt and the arrowhead shows the cleaved products (∼90 kDa). The second panel shows the level of Bcl-xL. IKKγ was used as loading control.

Techniques Used: Mutagenesis, Western Blot

Differentiation of MESC2.10 neurons and etoposide-induced DNA damage. (A) Neuroblasts were transduced with an EGFP lentivirus. After differentiation for 9 days they were fixed and examined by a confocal microscope. TOTO-3 was used to stain the nuclei. (B) Synapse markers in MESC2.10 neurons. Extracts from differentiated neurons (DPD, days post differentiation) were examined by Western blotting for expression of β-catenin and PSD-95. Bottom panel shows Western blot analysis of lysates from neurons maintained for different time points in culture stained for caspase-3 activation. (C) γ-H2aX accumulates in the nuclei of etoposide treated MESC2.10 neuron. Differentiated neurons treated with etoposide for 4hr were fixed and stained with a rabbit anti-γ-H2aX (green). Anti-Tuj-1 was used to label the cytoplasm (red). Part (D) shows accumulation of γ-H2aX in the nuclear fraction of etoposide treated neurons over time examined by Western blotting with anti-γ-H2aX antibody. Lamin B1 was used as loading control.
Figure Legend Snippet: Differentiation of MESC2.10 neurons and etoposide-induced DNA damage. (A) Neuroblasts were transduced with an EGFP lentivirus. After differentiation for 9 days they were fixed and examined by a confocal microscope. TOTO-3 was used to stain the nuclei. (B) Synapse markers in MESC2.10 neurons. Extracts from differentiated neurons (DPD, days post differentiation) were examined by Western blotting for expression of β-catenin and PSD-95. Bottom panel shows Western blot analysis of lysates from neurons maintained for different time points in culture stained for caspase-3 activation. (C) γ-H2aX accumulates in the nuclei of etoposide treated MESC2.10 neuron. Differentiated neurons treated with etoposide for 4hr were fixed and stained with a rabbit anti-γ-H2aX (green). Anti-Tuj-1 was used to label the cytoplasm (red). Part (D) shows accumulation of γ-H2aX in the nuclear fraction of etoposide treated neurons over time examined by Western blotting with anti-γ-H2aX antibody. Lamin B1 was used as loading control.

Techniques Used: Transduction, Microscopy, Staining, Western Blot, Expressing, Activation Assay

Etoposide promotes reduction of Bcl-xL. (A) Extracts of control and etoposide-treated MESC2.10 neurons were examined for Bcl-xL by Western blotting. Neurons were treated with etoposide for 6 hrs. Top panel shows staining for Bcl-xL and the bottom panel indicates IKKγ as a loading control. Fold changes were normalized to the intensity of loading control, and compared to that of untreated control neurons (lane 5). (B) Bcl-xL expression prevents Htt proteolysis by etoposide. MESC2.10 neuroblasts were transduced with a lentivirus expressing Bcl-xL (Lanes 3 and 4) and treated with etoposide for 6 hrs. EGFP-lentivirus was used a control (C). Top panel Western shows blotting for Htt. Arrow indicates the full-length Htt and the arrowhead shows the cleaved Htt products. Second and third panels show staining for Bcl-xL and pro-caspase-3, respectively. IKKγ levels were used as a loading control. (C) IKKβ phosphorylates Bcl-xL. Active recombinant IKKα or IKKβ were tested for the ability to phosphorylate Bcl-xL. The kinase assay was performed as described in the M M with recombinant Bcl-xL as a substrate. Products were visualized by autoradiography. IκBα was used as a positive control substrate for IKKα. The top panel shows the kinase product (KA) and bottom panel shows the SDS-PAGE and coomassie-blue staining of the substrates use in kinase assays.
Figure Legend Snippet: Etoposide promotes reduction of Bcl-xL. (A) Extracts of control and etoposide-treated MESC2.10 neurons were examined for Bcl-xL by Western blotting. Neurons were treated with etoposide for 6 hrs. Top panel shows staining for Bcl-xL and the bottom panel indicates IKKγ as a loading control. Fold changes were normalized to the intensity of loading control, and compared to that of untreated control neurons (lane 5). (B) Bcl-xL expression prevents Htt proteolysis by etoposide. MESC2.10 neuroblasts were transduced with a lentivirus expressing Bcl-xL (Lanes 3 and 4) and treated with etoposide for 6 hrs. EGFP-lentivirus was used a control (C). Top panel Western shows blotting for Htt. Arrow indicates the full-length Htt and the arrowhead shows the cleaved Htt products. Second and third panels show staining for Bcl-xL and pro-caspase-3, respectively. IKKγ levels were used as a loading control. (C) IKKβ phosphorylates Bcl-xL. Active recombinant IKKα or IKKβ were tested for the ability to phosphorylate Bcl-xL. The kinase assay was performed as described in the M M with recombinant Bcl-xL as a substrate. Products were visualized by autoradiography. IκBα was used as a positive control substrate for IKKα. The top panel shows the kinase product (KA) and bottom panel shows the SDS-PAGE and coomassie-blue staining of the substrates use in kinase assays.

Techniques Used: Western Blot, Staining, Expressing, Transduction, Recombinant, Kinase Assay, Autoradiography, Positive Control, SDS Page

Etoposide promotes IKKβand inhibits IKKα. (A) Etoposide activates IKKβ. IKK complexes were immunoprecipitated with anti-IKKγ antibody coupled to protein G agarose beads and assayed for kinase activity using GST-IκBα and 32 P-γ-ATP. Products were examined by SDS-PAGE followed by autoradiography. The top panel shows kinase activity (KA) and the lower panel shows a Western blot for IKKβ of similar immunoprecipitated complexes. (B) IKKα is constitutively active in MESC2.10 neurons. Lysates were first treated with a combination of anti-IKKβ and IKKγ antibodies coupled to agarose beads to deplete IKKγ/IKKβ/IKKα complex. IKKα complexes were then immunoprecipitated with anti-IKKα antibody conjugated to protein G agarose beads and assayed for kinase activity as described in part A. The top panel shows IKKα activity and the bottom panel shows the Western blot for IKKα. Fold changes of IKK activity were quantified by measuring the band intensity using Image J, and compared to non-treated neurons.
Figure Legend Snippet: Etoposide promotes IKKβand inhibits IKKα. (A) Etoposide activates IKKβ. IKK complexes were immunoprecipitated with anti-IKKγ antibody coupled to protein G agarose beads and assayed for kinase activity using GST-IκBα and 32 P-γ-ATP. Products were examined by SDS-PAGE followed by autoradiography. The top panel shows kinase activity (KA) and the lower panel shows a Western blot for IKKβ of similar immunoprecipitated complexes. (B) IKKα is constitutively active in MESC2.10 neurons. Lysates were first treated with a combination of anti-IKKβ and IKKγ antibodies coupled to agarose beads to deplete IKKγ/IKKβ/IKKα complex. IKKα complexes were then immunoprecipitated with anti-IKKα antibody conjugated to protein G agarose beads and assayed for kinase activity as described in part A. The top panel shows IKKα activity and the bottom panel shows the Western blot for IKKα. Fold changes of IKK activity were quantified by measuring the band intensity using Image J, and compared to non-treated neurons.

Techniques Used: Immunoprecipitation, Activity Assay, SDS Page, Autoradiography, Western Blot

3) Product Images from "Etoposide damages female germ cells in the developing ovary"

Article Title: Etoposide damages female germ cells in the developing ovary

Journal: BMC Cancer

doi: 10.1186/s12885-016-2505-9

Effect of etoposide on different stages of follicle development. Number and health of primordial (PF), transitional (TRNS) and primary (PRIM) follicles was determined in cultured mouse ovaries exposed to etoposide prior to (fetal ovaries: Ai , Bi ); or after (neonatal ovaries: Aii , Bii ) follicle formation. Figure shows: follicle distribution ( Ai , Aii ); and the percentage of follicles assessed as unhealthy for each follicle stage ( Bi , Bii ). c Photomicrographs of haemotoxylin and eosin stained cultured fetal and neonatal ovary sections, comparing healthy and unhealthy primordial, transitional and primary follicles. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p
Figure Legend Snippet: Effect of etoposide on different stages of follicle development. Number and health of primordial (PF), transitional (TRNS) and primary (PRIM) follicles was determined in cultured mouse ovaries exposed to etoposide prior to (fetal ovaries: Ai , Bi ); or after (neonatal ovaries: Aii , Bii ) follicle formation. Figure shows: follicle distribution ( Ai , Aii ); and the percentage of follicles assessed as unhealthy for each follicle stage ( Bi , Bii ). c Photomicrographs of haemotoxylin and eosin stained cultured fetal and neonatal ovary sections, comparing healthy and unhealthy primordial, transitional and primary follicles. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p

Techniques Used: Cell Culture, Staining

Germ cells show impaired capability of forming follicles in the presence of etoposide. A: Photomicrographs of haemotoxylin and eosin stained E13.5 CD1 ovaries cultured for 6 days either in control medium ( Ai ) or in medium supplemented with the highest dose of etoposide (150 ng ml −1 ; Aii ). By day 6 of culture, there were significantly fewer germ cells remaining in etoposide-treated ovaries than in controls ( b ). Arrows denote germ cells within the ovary. Scale bars: both 50 μm. Bars denote mean ± SEM, n = 5 for controls, n = 6 for treatment group. Stars denote significant differences relative to control (*** p
Figure Legend Snippet: Germ cells show impaired capability of forming follicles in the presence of etoposide. A: Photomicrographs of haemotoxylin and eosin stained E13.5 CD1 ovaries cultured for 6 days either in control medium ( Ai ) or in medium supplemented with the highest dose of etoposide (150 ng ml −1 ; Aii ). By day 6 of culture, there were significantly fewer germ cells remaining in etoposide-treated ovaries than in controls ( b ). Arrows denote germ cells within the ovary. Scale bars: both 50 μm. Bars denote mean ± SEM, n = 5 for controls, n = 6 for treatment group. Stars denote significant differences relative to control (*** p

Techniques Used: Staining, Cell Culture

Etoposide primarily targets oocytes prior to follicle formation and granulosa cells after follicle formation. Follicles were categorised as unhealthy due to: unhealthy oocyte (OOC) only ( i ); unhealthy granulosa cells (GCs) only ( ii ); or unhealthy oocyte and granulosa cells (OOC + GCs) ( iii ), in fetal ( a ) or neonatal ( b ) mouse ovaries exposed to etoposide. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p
Figure Legend Snippet: Etoposide primarily targets oocytes prior to follicle formation and granulosa cells after follicle formation. Follicles were categorised as unhealthy due to: unhealthy oocyte (OOC) only ( i ); unhealthy granulosa cells (GCs) only ( ii ); or unhealthy oocyte and granulosa cells (OOC + GCs) ( iii ), in fetal ( a ) or neonatal ( b ) mouse ovaries exposed to etoposide. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p

Techniques Used:

Etoposide has a significant effect on follicle numbers and health on fetal, but not neonatal ovaries. a Photomicrographs of haemotoxylin and eosin stained sections from fetal ( Ai-iii ) and neonatal ( Aiv-vi ) ovaries treated with etoposide in vitro. Fetal mouse ovaries cultured for twelve days in control medium (DMSO only) ( Ai ); or in the presence of 50 ng ml −1 ( Aii ); or 150 ng ml −1 ( Aiii ) etoposide for the first six days of culture. Neonatal mouse ovaries cultured for six days with control medium (DMSO only) (Aiv); or in the presence of 50 ng ml −1 ( Av ); or 150 ng ml −1 ( Avi ) etoposide for the six days of culture. b , c Follicle number ( b ) and health ( c ) was determined in cultured mouse ovaries exposed to etoposide prior to (fetal ovaries: Bi , Ci ) or after (neonatal ovaries: Bii , Cii ) follicle formation. Figure shows: total follicle numbers ( Bi , Bii ); and the percentage of follicles assessed as unhealthy ( Ci , Cii ). Red arrows show examples of follicles deemed unhealthy due to unhealthy oocyte; yellow arrow shows examples of follicles deemed unhealthy due to unhealthy granulosa cells. Scale bars 50 μm. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p
Figure Legend Snippet: Etoposide has a significant effect on follicle numbers and health on fetal, but not neonatal ovaries. a Photomicrographs of haemotoxylin and eosin stained sections from fetal ( Ai-iii ) and neonatal ( Aiv-vi ) ovaries treated with etoposide in vitro. Fetal mouse ovaries cultured for twelve days in control medium (DMSO only) ( Ai ); or in the presence of 50 ng ml −1 ( Aii ); or 150 ng ml −1 ( Aiii ) etoposide for the first six days of culture. Neonatal mouse ovaries cultured for six days with control medium (DMSO only) (Aiv); or in the presence of 50 ng ml −1 ( Av ); or 150 ng ml −1 ( Avi ) etoposide for the six days of culture. b , c Follicle number ( b ) and health ( c ) was determined in cultured mouse ovaries exposed to etoposide prior to (fetal ovaries: Bi , Ci ) or after (neonatal ovaries: Bii , Cii ) follicle formation. Figure shows: total follicle numbers ( Bi , Bii ); and the percentage of follicles assessed as unhealthy ( Ci , Cii ). Red arrows show examples of follicles deemed unhealthy due to unhealthy oocyte; yellow arrow shows examples of follicles deemed unhealthy due to unhealthy granulosa cells. Scale bars 50 μm. Bars denote mean ± SEM; n = 6 for all groups. Stars denote significant differences relative to control (* p

Techniques Used: Staining, In Vitro, Cell Culture

Oocytes from cultured fetal ovaries progress through prophase I of meiosis in the presence or absence of etoposide. Fetal ovaries cultured for 2, 4 or 6 days were stained for Sycp3 in order to assess the synaptonemal complex (SC). Oocytes were categorised into leptotene/zygotene, (SC assembling but not fully formed) by the presence of extensive networks of fine Sycp3 threads, often with large nuclear aggregates of Sycp3 that has not yet assembled into SC ( Ai ); pachytene, (fully synapsed SC) by the presence of a thicker well-spaced long Sycp3 threads ( Aii ); or diplotene, (SC disassembling but still present) by the presence of short thick fragments of Sycp3 threads ( Aiii ). B: Meiotic progression was examined in germ cells from control ovaries and from ovaries exposed to 150 ng ml −1 etoposide during culture. Control oocytes progressed through the early stages of prophase I in a normal manner during the first 6 days of culture, with the majority at leptotene/zygotene at Day 2 of culture, pachytene at Day 4 and diplotene by Day 6 of culture, immediately prior to follicle formation. Oocytes exposed to 150 ng ml −1 etoposide during culture were able to progress through to diplotene. At Day 2 of culture only, oocytes from etoposide-treated ovaries were at more advanced meiotic stages than those from control ovaries, but there was no difference at Days 4 or 6. Scale bars: 10 μm, n = 961 control oocytes and n = 994 treatment oocytes. Stars denote significant difference between oocytes from etoposide-exposed ovaries ( lower panel ) relative to controls ( upper panel ; ** p
Figure Legend Snippet: Oocytes from cultured fetal ovaries progress through prophase I of meiosis in the presence or absence of etoposide. Fetal ovaries cultured for 2, 4 or 6 days were stained for Sycp3 in order to assess the synaptonemal complex (SC). Oocytes were categorised into leptotene/zygotene, (SC assembling but not fully formed) by the presence of extensive networks of fine Sycp3 threads, often with large nuclear aggregates of Sycp3 that has not yet assembled into SC ( Ai ); pachytene, (fully synapsed SC) by the presence of a thicker well-spaced long Sycp3 threads ( Aii ); or diplotene, (SC disassembling but still present) by the presence of short thick fragments of Sycp3 threads ( Aiii ). B: Meiotic progression was examined in germ cells from control ovaries and from ovaries exposed to 150 ng ml −1 etoposide during culture. Control oocytes progressed through the early stages of prophase I in a normal manner during the first 6 days of culture, with the majority at leptotene/zygotene at Day 2 of culture, pachytene at Day 4 and diplotene by Day 6 of culture, immediately prior to follicle formation. Oocytes exposed to 150 ng ml −1 etoposide during culture were able to progress through to diplotene. At Day 2 of culture only, oocytes from etoposide-treated ovaries were at more advanced meiotic stages than those from control ovaries, but there was no difference at Days 4 or 6. Scale bars: 10 μm, n = 961 control oocytes and n = 994 treatment oocytes. Stars denote significant difference between oocytes from etoposide-exposed ovaries ( lower panel ) relative to controls ( upper panel ; ** p

Techniques Used: Cell Culture, Staining

4) Product Images from "EWS–FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma"

Article Title: EWS–FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma

Journal: Nature

doi: 10.1038/nature25748

R-loop accumulation in Ewing sarcoma. a , b , Fold-difference in genomic R-loops in IMR90 versus Ewing sarcoma cells ( a ) and U2OS cells with indicated transfections ( b ); n = 4 technical replicates. c , Representative immunofluorescence images of nuclei (DAPI), R-loops (S9.6) and nucleoli (nucleolin). Scale bar, 25 μm. d , Thirty-five-kilobase region surrounding the SON gene containing RNA-seq (red), RNAPII ChIP–seq (blue), DRIP–seq (black) and RNaseH-treated (RNH) tracks. Etop, etoposide-treated. Track height represents read counts. e , Immunoblots of indicated replication stress proteins. f , Cell viability response to ATR inhibitor; n = 4 technical replicates. g , Proliferation rate of TC32 cells overexpressing RNaseH1 (RNH1) after ATR inhibition (ATRi). EV, empty vector; n = 3 transfection replicates. Mean ± s.e.m., one-way ANOVA compared to IMR90 control, two-tailed t -test within each cell line. #,* P
Figure Legend Snippet: R-loop accumulation in Ewing sarcoma. a , b , Fold-difference in genomic R-loops in IMR90 versus Ewing sarcoma cells ( a ) and U2OS cells with indicated transfections ( b ); n = 4 technical replicates. c , Representative immunofluorescence images of nuclei (DAPI), R-loops (S9.6) and nucleoli (nucleolin). Scale bar, 25 μm. d , Thirty-five-kilobase region surrounding the SON gene containing RNA-seq (red), RNAPII ChIP–seq (blue), DRIP–seq (black) and RNaseH-treated (RNH) tracks. Etop, etoposide-treated. Track height represents read counts. e , Immunoblots of indicated replication stress proteins. f , Cell viability response to ATR inhibitor; n = 4 technical replicates. g , Proliferation rate of TC32 cells overexpressing RNaseH1 (RNH1) after ATR inhibition (ATRi). EV, empty vector; n = 3 transfection replicates. Mean ± s.e.m., one-way ANOVA compared to IMR90 control, two-tailed t -test within each cell line. #,* P

Techniques Used: Transfection, Immunofluorescence, RNA Sequencing Assay, Chromatin Immunoprecipitation, Western Blot, Inhibition, Plasmid Preparation, Two Tailed Test

Similarity of Ewing sarcoma to BRCA-deficient tumours. a , IC 50 levels of olaparib in EWS–FLI1 mutant cells ( n = 17) versus breast cancers ( n = 13) or pan-cancer ( n = 147) dataset. b , Cell viability of IMR90 and Ewing sarcoma cells with increasing doses of olaparib. Mean ± s.d., n = 3 technical replicates, one-way ANOVA compared to IMR90 cells. c , Cell viability plot demonstrating the role of EWS–FLI1 in mediating exquisite sensitivity to olaparib in U2OS cells transfected with either the oncogene or empty vector; n = 3 transfection replicates. d , Immunoblots depicting transfection efficiency of indicated siRNA and expression constructs used in . e , TP53BP1 knockdown improved Ewing sarcoma (TC32 cell) survival in response to damage. Immunoblots depict level of TP53BP1 knockdown. n = 4 transfection replicates. f , Representative immunoblots showing equivalent levels of BRCA1 in whole cell lysates (upper panel) from control and Ewing sarcoma cells with and without etoposide treatment (2 h). The lower panel shows BRCA1 redistribution in subcellular fractions of U2OS or TC32 cells. GAPDH and lamin B1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. g , Immunoblots of whole cell lysates and subcellular fractions from U2OS cells with and without EWSR1 depletion. Data indicated no change in BRCA1 levels with EWSR1 knockdown. Loading controls include: GAPDH for cytoplasm, Sp1 for nuclei and histone H3 for chromatin. Mean ± s.e.m., ** P
Figure Legend Snippet: Similarity of Ewing sarcoma to BRCA-deficient tumours. a , IC 50 levels of olaparib in EWS–FLI1 mutant cells ( n = 17) versus breast cancers ( n = 13) or pan-cancer ( n = 147) dataset. b , Cell viability of IMR90 and Ewing sarcoma cells with increasing doses of olaparib. Mean ± s.d., n = 3 technical replicates, one-way ANOVA compared to IMR90 cells. c , Cell viability plot demonstrating the role of EWS–FLI1 in mediating exquisite sensitivity to olaparib in U2OS cells transfected with either the oncogene or empty vector; n = 3 transfection replicates. d , Immunoblots depicting transfection efficiency of indicated siRNA and expression constructs used in . e , TP53BP1 knockdown improved Ewing sarcoma (TC32 cell) survival in response to damage. Immunoblots depict level of TP53BP1 knockdown. n = 4 transfection replicates. f , Representative immunoblots showing equivalent levels of BRCA1 in whole cell lysates (upper panel) from control and Ewing sarcoma cells with and without etoposide treatment (2 h). The lower panel shows BRCA1 redistribution in subcellular fractions of U2OS or TC32 cells. GAPDH and lamin B1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. g , Immunoblots of whole cell lysates and subcellular fractions from U2OS cells with and without EWSR1 depletion. Data indicated no change in BRCA1 levels with EWSR1 knockdown. Loading controls include: GAPDH for cytoplasm, Sp1 for nuclei and histone H3 for chromatin. Mean ± s.e.m., ** P

Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Expressing, Construct

Characterizing Ewing sarcoma chemosensitivity. a , Cell lines used in the study. b , Level of cell death caused by EWS–FLI1 knockdown alone in TC32 cells. Immunoblot shows extent of knockdown; n = 4 transfection replicates. c , Cell viability of U2OS cells transfected either with empty vector (EV) or EWS–FLI1 for 24 h before etoposide exposure for a further 48 h. Immunoblot shows transfection efficiency; n = 3 transfection replicates. d , IC 50 levels of etoposide or mitomycin in EWS–FLI1 mutant ( n = 16) versus pan-cancer ( n = 143) dataset. Brown lines, range of screening concentrations of the drug. Red lines, geometric mean of drug concentration. e , Heatmap of basal gene expression profile in control and Ewing sarcoma cell lines after hierarchical clustering. f , g , Top enriched pathways from gene set enrichment analysis of the differences between Ewing sarcoma and IMR90 cells are listed ( f ) and relevant signature plots are illustrated ( g ). We found differential upregulation of replication stress, BRCA1-mutation driven network and altered transcription regulation pathways in Ewing sarcoma. h , Cross-screen pathway comparison of top survival hits from RNAi screens in Drosophila Kc167 cells exposed to MMS, bleomycin or etoposide. Nearly a third of the top 5% hits in each screen were genes involved in transcription and RNA metabolism, highlighting the importance of this pathway in DNA damage survival. Mean ± s.e.m., * P
Figure Legend Snippet: Characterizing Ewing sarcoma chemosensitivity. a , Cell lines used in the study. b , Level of cell death caused by EWS–FLI1 knockdown alone in TC32 cells. Immunoblot shows extent of knockdown; n = 4 transfection replicates. c , Cell viability of U2OS cells transfected either with empty vector (EV) or EWS–FLI1 for 24 h before etoposide exposure for a further 48 h. Immunoblot shows transfection efficiency; n = 3 transfection replicates. d , IC 50 levels of etoposide or mitomycin in EWS–FLI1 mutant ( n = 16) versus pan-cancer ( n = 143) dataset. Brown lines, range of screening concentrations of the drug. Red lines, geometric mean of drug concentration. e , Heatmap of basal gene expression profile in control and Ewing sarcoma cell lines after hierarchical clustering. f , g , Top enriched pathways from gene set enrichment analysis of the differences between Ewing sarcoma and IMR90 cells are listed ( f ) and relevant signature plots are illustrated ( g ). We found differential upregulation of replication stress, BRCA1-mutation driven network and altered transcription regulation pathways in Ewing sarcoma. h , Cross-screen pathway comparison of top survival hits from RNAi screens in Drosophila Kc167 cells exposed to MMS, bleomycin or etoposide. Nearly a third of the top 5% hits in each screen were genes involved in transcription and RNA metabolism, highlighting the importance of this pathway in DNA damage survival. Mean ± s.e.m., * P

Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Concentration Assay, Expressing

R-loops in Ewing sarcoma. a , Quantification of RNA–DNA hybrids in TC32 cells transfected with either empty vector (EV) or RNaseH1 (RNH1). The immunoblot to the right indicates RNaseH1 transfection efficiency; n = 4 transfection replicates. b , RNA–DNA hybrid levels in TC32 cells with scrambled (siCtrl) or EWS–FLI1 (siFLI1) knockdown; n = 3 transfection replicates. c , Schematic of the EWS–FLI1 R2L2 construct. Arginine residues 383 and 386 (black bars) in EWS–FLI1 are converted to leucine to render the fusion oncogene deficient in DNA binding. Below is a quantification of RNA–DNA hybrids in U2OS cells expressing empty vector, EWS–FLI1 or EWS–FLI1 R2L2; n = 4 transfection replicates. d , e , Fold change in RNA–DNA hybrids after damage (etoposide, 6 h) in IMR90 versus Ewing sarcoma cells ( d ) or U2OS cells with either EWSR1 depletion or EWS–FLI1 expression ( e ). NT, no treatment; n = 4 technical or transfection replicates. f , Quantification of nucleoplasmic RNA–DNA hybrids in the immunofluorescence images ( n = 80 nuclei) demonstrated a clear increase in overall R-loop intensity in Ewing sarcoma cells. Nucleolin signal was used to subtract nucleolar R-loops. One-way ANOVA. Mean ± s.e.m., #,* P
Figure Legend Snippet: R-loops in Ewing sarcoma. a , Quantification of RNA–DNA hybrids in TC32 cells transfected with either empty vector (EV) or RNaseH1 (RNH1). The immunoblot to the right indicates RNaseH1 transfection efficiency; n = 4 transfection replicates. b , RNA–DNA hybrid levels in TC32 cells with scrambled (siCtrl) or EWS–FLI1 (siFLI1) knockdown; n = 3 transfection replicates. c , Schematic of the EWS–FLI1 R2L2 construct. Arginine residues 383 and 386 (black bars) in EWS–FLI1 are converted to leucine to render the fusion oncogene deficient in DNA binding. Below is a quantification of RNA–DNA hybrids in U2OS cells expressing empty vector, EWS–FLI1 or EWS–FLI1 R2L2; n = 4 transfection replicates. d , e , Fold change in RNA–DNA hybrids after damage (etoposide, 6 h) in IMR90 versus Ewing sarcoma cells ( d ) or U2OS cells with either EWSR1 depletion or EWS–FLI1 expression ( e ). NT, no treatment; n = 4 technical or transfection replicates. f , Quantification of nucleoplasmic RNA–DNA hybrids in the immunofluorescence images ( n = 80 nuclei) demonstrated a clear increase in overall R-loop intensity in Ewing sarcoma cells. Nucleolin signal was used to subtract nucleolar R-loops. One-way ANOVA. Mean ± s.e.m., #,* P

Techniques Used: Transfection, Plasmid Preparation, Construct, Binding Assay, Expressing, Immunofluorescence

Genome-wide heatmaps. a , Heatmaps representing genome-wide localization of RNAPII, BRCA1 and R-loop sites centred on the TSS. The data were sorted by DRIP sites. The upper panel represents untreated (NT) samples and the lower panel represents etoposide (Etop, 6 h) treated samples. There was a clear decrease in BRCA1 and R-loop signal upon damage in the control cell lines, unlike in Ewing sarcoma. b , KS plots to demonstrate empirical distribution of the top 13.8% of DRIP and ChIP peaks and higher expression relative to uniform distribution. Data are sorted by BRCA1 ChIP, n = 3,066 genes. c , P values of statistical comparisons between RNAPII ChIP and R-loop probability distributions for all cell lines against IMR90 DRIP data centred on the TSS. The top 27% of DRIP–seq peaks corresponding to 6,127 genes were used for the analysis and data were sorted by BRCA1 binding sites.
Figure Legend Snippet: Genome-wide heatmaps. a , Heatmaps representing genome-wide localization of RNAPII, BRCA1 and R-loop sites centred on the TSS. The data were sorted by DRIP sites. The upper panel represents untreated (NT) samples and the lower panel represents etoposide (Etop, 6 h) treated samples. There was a clear decrease in BRCA1 and R-loop signal upon damage in the control cell lines, unlike in Ewing sarcoma. b , KS plots to demonstrate empirical distribution of the top 13.8% of DRIP and ChIP peaks and higher expression relative to uniform distribution. Data are sorted by BRCA1 ChIP, n = 3,066 genes. c , P values of statistical comparisons between RNAPII ChIP and R-loop probability distributions for all cell lines against IMR90 DRIP data centred on the TSS. The top 27% of DRIP–seq peaks corresponding to 6,127 genes were used for the analysis and data were sorted by BRCA1 binding sites.

Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Expressing, Binding Assay

Ewing sarcoma dysregulates transcription in response to damage. a , Cell viability following etoposide treatment. Etoposide dose causing 35% lethality (LD35, dotted grey line) was used for further experiments. Mean ± s.d., n = 4 technical replicates, one-way ANOVA. b , Etoposide-induced TC32 cytotoxicity after EWS–FLI1 knockdown (siFLI1). n = 4 transfection replicates, two-tailed t -tests. c , Heatmap of damage-induced differential gene expression. d , CDK9 kinase activity inhibition by recombinant EWSR1 and FUS proteins on CDKtide or CTD substrates. n = 3 technical replicates, one-way ANOVA. e – g , Levels of phosphorylated Ser2/Ser5 RNAPII in U2OS cells with EWSR1 knockdown ( e ), IMR90 cells versus TC32 cells ( f ), and TC32 cells with EWS–FLI1 knockdown ( g ). h , Transcriptional activity after etoposide treatment. Centre at median, n = 100 cells, two-way ANOVA. Mean ± s.e.m., * P
Figure Legend Snippet: Ewing sarcoma dysregulates transcription in response to damage. a , Cell viability following etoposide treatment. Etoposide dose causing 35% lethality (LD35, dotted grey line) was used for further experiments. Mean ± s.d., n = 4 technical replicates, one-way ANOVA. b , Etoposide-induced TC32 cytotoxicity after EWS–FLI1 knockdown (siFLI1). n = 4 transfection replicates, two-tailed t -tests. c , Heatmap of damage-induced differential gene expression. d , CDK9 kinase activity inhibition by recombinant EWSR1 and FUS proteins on CDKtide or CTD substrates. n = 3 technical replicates, one-way ANOVA. e – g , Levels of phosphorylated Ser2/Ser5 RNAPII in U2OS cells with EWSR1 knockdown ( e ), IMR90 cells versus TC32 cells ( f ), and TC32 cells with EWS–FLI1 knockdown ( g ). h , Transcriptional activity after etoposide treatment. Centre at median, n = 100 cells, two-way ANOVA. Mean ± s.e.m., * P

Techniques Used: Transfection, Two Tailed Test, Expressing, Activity Assay, Inhibition, Recombinant

Association of BRCA1 with the transcription complex in Ewing sarcoma. a , Co-immunoprecipitation: immunoblots of IMR90 and EWS502 nuclear lysates with and without exposure to etoposide (2 h). The left panel indicates 10% of the input used for immunoprecipitation. BRCA1 antibody was used for immunoprecipitation in the middle panel and the rightmost panel indicates specificity of interaction against IgG pulldown. b , Real-time qPCR analysis of BRCA ChIP samples from control and Ewing sarcoma cell lines with and without etoposide treatment, using primers within the FEN1 and PARP8 genes. c , Representative sequencing track image of gene expression (red tracks), R-loop sites (black and grey tracks) and BRCA1 binding sites (blue tracks) across the FEN1 gene demonstrating the enrichment of R-loops and BRCA1 in the region amplified by the primers in b . d , qPCR analysis as in b with primers targeting a well-known R-loop region within the APOE gene. e , Representative sequencing track image as in c across the APOE gene. f , Agarose gel blots evaluating amplicons generated using EWS502 DRIPs with primers against FEN1 and PARP8 . NT, no treatment; Etop, etoposide-treated (6 h); RNH, RNaseH-treated samples. Mean ± s.e.m., n = 3 technical replicates, ** P
Figure Legend Snippet: Association of BRCA1 with the transcription complex in Ewing sarcoma. a , Co-immunoprecipitation: immunoblots of IMR90 and EWS502 nuclear lysates with and without exposure to etoposide (2 h). The left panel indicates 10% of the input used for immunoprecipitation. BRCA1 antibody was used for immunoprecipitation in the middle panel and the rightmost panel indicates specificity of interaction against IgG pulldown. b , Real-time qPCR analysis of BRCA ChIP samples from control and Ewing sarcoma cell lines with and without etoposide treatment, using primers within the FEN1 and PARP8 genes. c , Representative sequencing track image of gene expression (red tracks), R-loop sites (black and grey tracks) and BRCA1 binding sites (blue tracks) across the FEN1 gene demonstrating the enrichment of R-loops and BRCA1 in the region amplified by the primers in b . d , qPCR analysis as in b with primers targeting a well-known R-loop region within the APOE gene. e , Representative sequencing track image as in c across the APOE gene. f , Agarose gel blots evaluating amplicons generated using EWS502 DRIPs with primers against FEN1 and PARP8 . NT, no treatment; Etop, etoposide-treated (6 h); RNH, RNaseH-treated samples. Mean ± s.e.m., n = 3 technical replicates, ** P

Techniques Used: Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Sequencing, Expressing, Binding Assay, Amplification, Agarose Gel Electrophoresis, Generated

5) Product Images from "Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations"

Article Title: Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations

Journal: eLife

doi: 10.7554/eLife.32222

Establishment of stable and inducible ARP8 expressing 11–4 Flp-In cells. ( A ) Immunofluorescence staining of 11–4 Flp-In cells expressing HA-tagged wild-type ARP8 (WT) or S412 phospho-deficient ARP8 (SA). For induction of HA-ARP8 expression, tetracycline was added for 24 hr. The anti-HA antibody was used to identify the expression of HA-ARP8. DAPI was used for DNA staining. ( B and C ) Immunoblotting analysis of phosphorylated-ATM, and expression level of INO80 and RAD51 in 11–4 Flp-In cells. The endogenous ARP8-depleted11-4 Flp-In cells with tetracycline inducible expression of siRNA-resistant wild-type (WT) ( B ) or S412A ARP8 (S412A) ( C ), were treated with/without 10 μM ATM inhibitor for 2 hr, before DMSO (ctrl) or etoposide treatment. The cells were harvested at the indicated time point after etoposide removal. β-actin is shown as a loading control.
Figure Legend Snippet: Establishment of stable and inducible ARP8 expressing 11–4 Flp-In cells. ( A ) Immunofluorescence staining of 11–4 Flp-In cells expressing HA-tagged wild-type ARP8 (WT) or S412 phospho-deficient ARP8 (SA). For induction of HA-ARP8 expression, tetracycline was added for 24 hr. The anti-HA antibody was used to identify the expression of HA-ARP8. DAPI was used for DNA staining. ( B and C ) Immunoblotting analysis of phosphorylated-ATM, and expression level of INO80 and RAD51 in 11–4 Flp-In cells. The endogenous ARP8-depleted11-4 Flp-In cells with tetracycline inducible expression of siRNA-resistant wild-type (WT) ( B ) or S412A ARP8 (S412A) ( C ), were treated with/without 10 μM ATM inhibitor for 2 hr, before DMSO (ctrl) or etoposide treatment. The cells were harvested at the indicated time point after etoposide removal. β-actin is shown as a loading control.

Techniques Used: Expressing, Immunofluorescence, Staining

Dual-color FISH analysis of chromosome 11q23 using a different DNA probe set. ( A ) Dual-color FISH analysis of chromosome 11q23 in AT5BIVA and 11–4 cells, using the probe from Vysis. The non-targeting control siRNA (siNT) or siARP8-depleted cells were treated with etoposide for 15 min, washed, and cultured for 6 hr in fresh medium. Values represent the means ± SE from three independent experiments. *p
Figure Legend Snippet: Dual-color FISH analysis of chromosome 11q23 using a different DNA probe set. ( A ) Dual-color FISH analysis of chromosome 11q23 in AT5BIVA and 11–4 cells, using the probe from Vysis. The non-targeting control siRNA (siNT) or siARP8-depleted cells were treated with etoposide for 15 min, washed, and cultured for 6 hr in fresh medium. Values represent the means ± SE from three independent experiments. *p

Techniques Used: Fluorescence In Situ Hybridization, Cell Culture

ARP8 contains an ATM/ATR substrate SQ motif at Ser412 and Q413. ( A ) Damage-induced INO80 phosphorylation was not detected by an ATM/ATR antibody. Immunoprecipitation analysis of INO80 phosphorylation. After treatment with or without etoposide for 15 min, cells were cultured in fresh medium for 0.5, 1 or 2 hr. The whole cell l extracts from U2OS cells were incubated with anti-INO80 or normal IgG-conjugated Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-ATM/ATR substrate antibody or an anti-INO80 antibody. The arrows indicate the position of INO80. ( B ) Schematic diagram of full-length human ARP8. The insertions of ARP8 are shown in yellow, and the SQ motif in insertion IV is pink. ( C ) A 3D structure model of ARP8. Insertion I, insertion IV, and the locations of important amino acid residues are indicated. This structure was obtained from the Protein Database (PDB ID: 4FO0), and was modified according to the report by Gerhold et al. and the description for PDB ID: 4FO0. ( D ) Alignment of multiple ARP8 sequences. The partial amino acid sequences containing the SQ motif or the corresponding amino acids, which are colored red. The homologous sequence corresponding to human ARP8 was obtained from the NCBI Protein Database.
Figure Legend Snippet: ARP8 contains an ATM/ATR substrate SQ motif at Ser412 and Q413. ( A ) Damage-induced INO80 phosphorylation was not detected by an ATM/ATR antibody. Immunoprecipitation analysis of INO80 phosphorylation. After treatment with or without etoposide for 15 min, cells were cultured in fresh medium for 0.5, 1 or 2 hr. The whole cell l extracts from U2OS cells were incubated with anti-INO80 or normal IgG-conjugated Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-ATM/ATR substrate antibody or an anti-INO80 antibody. The arrows indicate the position of INO80. ( B ) Schematic diagram of full-length human ARP8. The insertions of ARP8 are shown in yellow, and the SQ motif in insertion IV is pink. ( C ) A 3D structure model of ARP8. Insertion I, insertion IV, and the locations of important amino acid residues are indicated. This structure was obtained from the Protein Database (PDB ID: 4FO0), and was modified according to the report by Gerhold et al. and the description for PDB ID: 4FO0. ( D ) Alignment of multiple ARP8 sequences. The partial amino acid sequences containing the SQ motif or the corresponding amino acids, which are colored red. The homologous sequence corresponding to human ARP8 was obtained from the NCBI Protein Database.

Techniques Used: Immunoprecipitation, Cell Culture, Incubation, Western Blot, Modification, Sequencing

ATM, but not ATR, negatively regulates RAD51 loading onto the BCR after etoposide treatment to repress 11q23 chromosome translocations. ( A ) ChIP analysis of the RAD51 loading onto the MLL BCR in ATMi or ATRi or a combination of ATMi and ATRi treated 11–4 cells. 11–4 cells were treated with ATMi (10 µM), ATRi (10 µM), or a combination of ATMi and ATRi for 2 hr before etoposide treatment. After washing the cells, the inhibitors (5 μM) were added until the cells were harvested. The ChIP analysis was performed as described in Figure 4 . Values represent the means ± SE from three independent experiments. *p
Figure Legend Snippet: ATM, but not ATR, negatively regulates RAD51 loading onto the BCR after etoposide treatment to repress 11q23 chromosome translocations. ( A ) ChIP analysis of the RAD51 loading onto the MLL BCR in ATMi or ATRi or a combination of ATMi and ATRi treated 11–4 cells. 11–4 cells were treated with ATMi (10 µM), ATRi (10 µM), or a combination of ATMi and ATRi for 2 hr before etoposide treatment. After washing the cells, the inhibitors (5 μM) were added until the cells were harvested. The ChIP analysis was performed as described in Figure 4 . Values represent the means ± SE from three independent experiments. *p

Techniques Used: Chromatin Immunoprecipitation

( A ) Etoposide treatment induced H2AX phosphorylation on BCR of MLL gene. ChIP analysis of γH2AX enrichment on the MLL BCR in ATM proficient 11–4 cells. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then fixed immediately (0 hr) or after culture in fresh medium for 0.5 hr (0.5 hr). β-globin (betaG) is shown as the control region. Values represent the means ± SE from four independent experiments. The results of immunoblotting analyses of γH2AX and H2AX are shown. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then harvested immediately (0 hr) or after culture in fresh medium for 0 (Etp) or 0.5 hr (0.5 hr). Source data are presented in Figure 2—source data 2 . ( B ) Depletion of ARP8 reduced the etoposide-induced enrichment of INO80 onto the BCR of the MLL gene. ChIP analysis of INO80 loading onto the MLL BCR in AT5BIVA cells depleted by either a non-targeting siRNA (siNT) or siARP8. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then cultured in fresh medium for 30 or 60 min. GAPDH is shown as the control region. Values represent the means ± SE from four independent experiments. *: p
Figure Legend Snippet: ( A ) Etoposide treatment induced H2AX phosphorylation on BCR of MLL gene. ChIP analysis of γH2AX enrichment on the MLL BCR in ATM proficient 11–4 cells. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then fixed immediately (0 hr) or after culture in fresh medium for 0.5 hr (0.5 hr). β-globin (betaG) is shown as the control region. Values represent the means ± SE from four independent experiments. The results of immunoblotting analyses of γH2AX and H2AX are shown. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then harvested immediately (0 hr) or after culture in fresh medium for 0 (Etp) or 0.5 hr (0.5 hr). Source data are presented in Figure 2—source data 2 . ( B ) Depletion of ARP8 reduced the etoposide-induced enrichment of INO80 onto the BCR of the MLL gene. ChIP analysis of INO80 loading onto the MLL BCR in AT5BIVA cells depleted by either a non-targeting siRNA (siNT) or siARP8. The cells were treated with DMSO (ctrl) or etoposide for 15 min, washed, and then cultured in fresh medium for 30 or 60 min. GAPDH is shown as the control region. Values represent the means ± SE from four independent experiments. *: p

Techniques Used: Chromatin Immunoprecipitation, Cell Culture

Examination of the interaction between ARP8 and INO80. ( A ) Examination of the interaction between INO80 and wild-type ARP8 or phospho-deficient ARP8. U2OS cells were co-transfected with Flag-INO80 and HA (vet), HA-ARP8 (WT), or HA-ARP8 S412A vectors, respectively, for 48 hr and subsequently treated with DMSO (ctrl) or etoposide for 15 min, washed, and then cultured in fresh medium for 2 hr. The immunoprecipitation analysis was performed using anti-HA antibodies. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-Flag or an anti-HA or anti-ATM/ATR substrate antibody. The co-immunoprecipitated amounts of Flag-INO80 relative to HA-ARP8 are shown. Quantitative analysis was performed using the Image J software. ( B ) Co-immunoprecipitation analysis of ARP8 and INO80. ATM-deficient BIVA cells were co-transfected with the siARP8 and siARP8-resistant HA-tagged wild-type ARP8 or phospho-mimetic S412D ARP8 mutant. After etoposide removal, the cells were recovered at the indicated time points. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with either an anti-INO80 antibody or an anti-HA antibody. The amounts of INO80 and HA-ARP8 were quantified, using the Image J software. The results of quantitative analysis are shown as the relative values as compared to the DMSO control, from three independent experiments. Source data are presented in Figure 3—source data 2 .
Figure Legend Snippet: Examination of the interaction between ARP8 and INO80. ( A ) Examination of the interaction between INO80 and wild-type ARP8 or phospho-deficient ARP8. U2OS cells were co-transfected with Flag-INO80 and HA (vet), HA-ARP8 (WT), or HA-ARP8 S412A vectors, respectively, for 48 hr and subsequently treated with DMSO (ctrl) or etoposide for 15 min, washed, and then cultured in fresh medium for 2 hr. The immunoprecipitation analysis was performed using anti-HA antibodies. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-Flag or an anti-HA or anti-ATM/ATR substrate antibody. The co-immunoprecipitated amounts of Flag-INO80 relative to HA-ARP8 are shown. Quantitative analysis was performed using the Image J software. ( B ) Co-immunoprecipitation analysis of ARP8 and INO80. ATM-deficient BIVA cells were co-transfected with the siARP8 and siARP8-resistant HA-tagged wild-type ARP8 or phospho-mimetic S412D ARP8 mutant. After etoposide removal, the cells were recovered at the indicated time points. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with either an anti-INO80 antibody or an anti-HA antibody. The amounts of INO80 and HA-ARP8 were quantified, using the Image J software. The results of quantitative analysis are shown as the relative values as compared to the DMSO control, from three independent experiments. Source data are presented in Figure 3—source data 2 .

Techniques Used: Transfection, Cell Culture, Immunoprecipitation, Western Blot, Software, Mutagenesis, Incubation

Requirement of INO80 and ARP8 for RAD51 binding to the BCR of the MLL gene. ( A ) Immunoblotting analysis of INO80 depletion efficiency. Two kinds of siRNAs against INO80 were used for the detection of the INO80 knockdown efficiency. RAD51 expression did not affect the INO80 knockdown. β−actin was used as the loading control. ( B ) ChIP analysis of the enrichment of RAD51 in AT5BIVA cells treated with non-targeting siRNA (siNT) or the siRNAs against INO80 (siINO80-1 and siINO80-4). The cells were treated with etoposide for 15 min, washed, and then cultured in fresh medium for 1 hr (Etp). Vehicle treatment was performed as a control. The DNA was analyzed by real-time PCR, using the MLL BCR gene primers. The β−globin gene (betaG) was used as a control region. Values represent the means ± SE from six independent experiments. *p
Figure Legend Snippet: Requirement of INO80 and ARP8 for RAD51 binding to the BCR of the MLL gene. ( A ) Immunoblotting analysis of INO80 depletion efficiency. Two kinds of siRNAs against INO80 were used for the detection of the INO80 knockdown efficiency. RAD51 expression did not affect the INO80 knockdown. β−actin was used as the loading control. ( B ) ChIP analysis of the enrichment of RAD51 in AT5BIVA cells treated with non-targeting siRNA (siNT) or the siRNAs against INO80 (siINO80-1 and siINO80-4). The cells were treated with etoposide for 15 min, washed, and then cultured in fresh medium for 1 hr (Etp). Vehicle treatment was performed as a control. The DNA was analyzed by real-time PCR, using the MLL BCR gene primers. The β−globin gene (betaG) was used as a control region. Values represent the means ± SE from six independent experiments. *p

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction

ATM, but not CK2 is responsible for ARP8 phosphorylation after etoposide treatment. ( A ) Immunoprecipitation analysis of U2OS cells transiently expressing HA-tagged ARP8, treated with or without an ATM inhibitor (ATMi). ATM inhibitor (10 or 20 μM) was added as indicated for 2 hr before etoposide (Etp) treatment. ( B ) Immunoprecipitation analysis of ARP8 phosphorylation, using U2OS cells cotransfected with HA-tagged ARP8 and either siATM, siCK2, or the double siRNAs (dKD) for 48 hr. The cells were cultured for 2 hr in fresh medium after etoposide (Etp) treatment. Immunoprecipitation was performed with anti-HA-conjugated beads. The precipitated proteins were detected by western blotting, using the indicated antibodies.
Figure Legend Snippet: ATM, but not CK2 is responsible for ARP8 phosphorylation after etoposide treatment. ( A ) Immunoprecipitation analysis of U2OS cells transiently expressing HA-tagged ARP8, treated with or without an ATM inhibitor (ATMi). ATM inhibitor (10 or 20 μM) was added as indicated for 2 hr before etoposide (Etp) treatment. ( B ) Immunoprecipitation analysis of ARP8 phosphorylation, using U2OS cells cotransfected with HA-tagged ARP8 and either siATM, siCK2, or the double siRNAs (dKD) for 48 hr. The cells were cultured for 2 hr in fresh medium after etoposide (Etp) treatment. Immunoprecipitation was performed with anti-HA-conjugated beads. The precipitated proteins were detected by western blotting, using the indicated antibodies.

Techniques Used: Immunoprecipitation, Expressing, Cell Culture, Western Blot

6) Product Images from "FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation"

Article Title: FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.404

Intracellular FGF1 WT protects SH-SY5Y cells from p53-dependent apoptosis. Stably transfected SH-SY5Y cells with either FGF1 WT or empty (mock) expression vectors were treated or not with etoposide for 16 h. ( a ) Polyclonal transfected SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the mock control, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) FGF1 expression and p53 activation (phosphorylated Ser15) were assessed by western blot in either polyclonal transfected cells (left panel) or isolated transfected cell lines (right panel). Actin detection was used as control. ( c ) FGF1 expression, caspase-9 and -3 cleavages were assessed in polyclonal transfected cells by western blot. Actin detection was used as control
Figure Legend Snippet: Intracellular FGF1 WT protects SH-SY5Y cells from p53-dependent apoptosis. Stably transfected SH-SY5Y cells with either FGF1 WT or empty (mock) expression vectors were treated or not with etoposide for 16 h. ( a ) Polyclonal transfected SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the mock control, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) FGF1 expression and p53 activation (phosphorylated Ser15) were assessed by western blot in either polyclonal transfected cells (left panel) or isolated transfected cell lines (right panel). Actin detection was used as control. ( c ) FGF1 expression, caspase-9 and -3 cleavages were assessed in polyclonal transfected cells by western blot. Actin detection was used as control

Techniques Used: Stable Transfection, Transfection, Expressing, Flow Cytometry, Cytometry, Staining, Activation Assay, Western Blot, Isolation

Intracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were transiently co-transfected with GFP and FGF1 WT or empty (mock) expression vectors and then treated or not with etoposide for 24 h. Following transfection and treatment, N2a transfected apoptotic cells were quantified by flow cytometry after CMX-Ros staining. Transfected apoptotic cells correspond to the high GFP (transfected cells, GFP+), low CMX-Ros (low ΔΨm, CMX−) and small-sized (size−) cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control Mock cells, except where indicated ( n =3; n.s.: P > 0.05; **: P ⩽0.01). ( b ) N2a cells were transiently transfected with FGF1 WT or empty (mock) expression vectors and then treated or not with etoposide for 24 h. FGF1 expression, p53 activation (Ser15 phosphorylation) and caspase-3 cleavage were analyzed by western blot. Actin detection was used as a control
Figure Legend Snippet: Intracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were transiently co-transfected with GFP and FGF1 WT or empty (mock) expression vectors and then treated or not with etoposide for 24 h. Following transfection and treatment, N2a transfected apoptotic cells were quantified by flow cytometry after CMX-Ros staining. Transfected apoptotic cells correspond to the high GFP (transfected cells, GFP+), low CMX-Ros (low ΔΨm, CMX−) and small-sized (size−) cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control Mock cells, except where indicated ( n =3; n.s.: P > 0.05; **: P ⩽0.01). ( b ) N2a cells were transiently transfected with FGF1 WT or empty (mock) expression vectors and then treated or not with etoposide for 24 h. FGF1 expression, p53 activation (Ser15 phosphorylation) and caspase-3 cleavage were analyzed by western blot. Actin detection was used as a control

Techniques Used: Transfection, Expressing, Flow Cytometry, Cytometry, Staining, Activation Assay, Western Blot

Extracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were pretreated or not by adding recombinant FGF1 and heparin in the culture medium (rFGF1) for 48 h, then treated or not with etoposide (Eto) for 24 h. N2a apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to control cells, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) N2a cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 24 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control
Figure Legend Snippet: Extracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were pretreated or not by adding recombinant FGF1 and heparin in the culture medium (rFGF1) for 48 h, then treated or not with etoposide (Eto) for 24 h. N2a apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to control cells, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) N2a cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 24 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

Techniques Used: Recombinant, Flow Cytometry, Cytometry, Staining, Western Blot, Activation Assay

Role of extra- and intracellular FGF1 in neuronal SH-SY5Y, N2a and PC12 cells. ( a ) Both extra- and intra-cellular FGF1 protect human SH-SY5Y and rat PC12 cells from p53-dependent apoptosis (left). Endogenous fgf1 expression is increased by rFGF1 addition (top left). In contrast, extra- and intra-cellular FGF1 show no anti-apoptotic activity, and rFGF1 addition does not increase endogenous fgf1 expression in murine N2a cells (right). ( b ) Overexpression of intracellular FGF1 inhibits etoposide-induced apoptosis in human SH-SY5Y and rat PC12 cells by decreasing PUMA transactivation, mitochondrial membrane depolarization and permeabilization, and activation of caspase-9, caspase-3 as assessed by the cleavage of its substrate PARP (left). On the contrary, FGF1 overexpression does not protect N2a cells from etoposide-induced apoptosis, and modifies neither p53 activation, mitochondrial depolarization and permeabilization, nor cleavage of caspase-3 (right). ( c ) In human SH-SY5Y and rat PC12 cells, overexpression of wild-type FGF1 or non-phosphorylable FGF1 S130A inhibits p53-dependent apoptosis, while overexpression of the phosphomimetic FGF1 S130D or the FGF1 K132E mutant does not. Therefore, phosphorylation of FGF1 inhibits its anti-apoptotic activity in SH-SY5Y and PC12 cells. This figure compiles the results of the present study performed in neuroblastoma SH-SY5Y and N2a cells and of previous studies performed in pheochromocytoma PC12 cells 14 , 15 , 32
Figure Legend Snippet: Role of extra- and intracellular FGF1 in neuronal SH-SY5Y, N2a and PC12 cells. ( a ) Both extra- and intra-cellular FGF1 protect human SH-SY5Y and rat PC12 cells from p53-dependent apoptosis (left). Endogenous fgf1 expression is increased by rFGF1 addition (top left). In contrast, extra- and intra-cellular FGF1 show no anti-apoptotic activity, and rFGF1 addition does not increase endogenous fgf1 expression in murine N2a cells (right). ( b ) Overexpression of intracellular FGF1 inhibits etoposide-induced apoptosis in human SH-SY5Y and rat PC12 cells by decreasing PUMA transactivation, mitochondrial membrane depolarization and permeabilization, and activation of caspase-9, caspase-3 as assessed by the cleavage of its substrate PARP (left). On the contrary, FGF1 overexpression does not protect N2a cells from etoposide-induced apoptosis, and modifies neither p53 activation, mitochondrial depolarization and permeabilization, nor cleavage of caspase-3 (right). ( c ) In human SH-SY5Y and rat PC12 cells, overexpression of wild-type FGF1 or non-phosphorylable FGF1 S130A inhibits p53-dependent apoptosis, while overexpression of the phosphomimetic FGF1 S130D or the FGF1 K132E mutant does not. Therefore, phosphorylation of FGF1 inhibits its anti-apoptotic activity in SH-SY5Y and PC12 cells. This figure compiles the results of the present study performed in neuroblastoma SH-SY5Y and N2a cells and of previous studies performed in pheochromocytoma PC12 cells 14 , 15 , 32

Techniques Used: Expressing, Activity Assay, Over Expression, Activation Assay, Mutagenesis

Extracellular FGF1 and etoposide increase endogenous fgf1 expression in SH-SY5Y cells, in contrast to N2a cells. SH-SY5Y and N2a cells were treated or not with rFGF1 for 72 h ( a – c ) or etoposide for 16 h ( b – d ). The levels of all fgf1 mRNAs ( a , b ) or of the alternative 1B fgf1 mRNA ( c , d ) were analyzed by RT-PCR. The 18S rRNA levels were used as a control for quantifications. The graphs represent the mean ±S.E.M. of three independent experiments. Student’s t -tests were performed ( n =3; n.s.: P > 0.05; * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001)
Figure Legend Snippet: Extracellular FGF1 and etoposide increase endogenous fgf1 expression in SH-SY5Y cells, in contrast to N2a cells. SH-SY5Y and N2a cells were treated or not with rFGF1 for 72 h ( a – c ) or etoposide for 16 h ( b – d ). The levels of all fgf1 mRNAs ( a , b ) or of the alternative 1B fgf1 mRNA ( c , d ) were analyzed by RT-PCR. The 18S rRNA levels were used as a control for quantifications. The graphs represent the mean ±S.E.M. of three independent experiments. Student’s t -tests were performed ( n =3; n.s.: P > 0.05; * P ⩽0.05; ** P ⩽0.01; *** P ⩽0.001)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

Extracellular FGF1 protects SH-SY5Y cells from p53-dependent apoptosis. ( a ) SH-SY5Y cells were pretreated or not by adding recombinant FGF1 and heparin for 48 h (rFGF1) in the culture medium, then cells were treated or not with etoposide for 24 h (Eto). Cell survival was analyzed by crystal violet nuclei staining. ( b ) Following the same treatments, SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells correspond to the low DiOC 6 (3) (low ΔΨm, noted DIOC−) and low PI (to exclude necrotic cells, noted PI−) staining and small-sized cells (a hallmark of apoptotic cell condensation, noted size−). For ( a and b ), the graphs represent the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control cells, except where indicated ( n =3; n.s.: P > 0.05; ** P ⩽0.01; *** P ⩽ 0.001). ( c ) SH-SY5Y cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 6 h or 17 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of the p53 proapoptotic target PUMA, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control
Figure Legend Snippet: Extracellular FGF1 protects SH-SY5Y cells from p53-dependent apoptosis. ( a ) SH-SY5Y cells were pretreated or not by adding recombinant FGF1 and heparin for 48 h (rFGF1) in the culture medium, then cells were treated or not with etoposide for 24 h (Eto). Cell survival was analyzed by crystal violet nuclei staining. ( b ) Following the same treatments, SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells correspond to the low DiOC 6 (3) (low ΔΨm, noted DIOC−) and low PI (to exclude necrotic cells, noted PI−) staining and small-sized cells (a hallmark of apoptotic cell condensation, noted size−). For ( a and b ), the graphs represent the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control cells, except where indicated ( n =3; n.s.: P > 0.05; ** P ⩽0.01; *** P ⩽ 0.001). ( c ) SH-SY5Y cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 6 h or 17 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of the p53 proapoptotic target PUMA, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

Techniques Used: Recombinant, Staining, Flow Cytometry, Cytometry, Western Blot, Activation Assay

FGF1 phosphorylation inhibits its anti-apoptotic activity in SH-SY5Y cells. SH-SY5Y cells were stably transfected with FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or empty (mock) expression vectors. ( a ) FGF1 levels were examined by western blot after heparin-sepharose concentration in cell lysates and conditioned media from SH-SY5Y cells expressing FGF1 WT , FGF1 S130A , FGF1 S130D or transfected with an empty vector (mock). ( b ) Apoptosis in FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or mock stably transfected SH-SY5Y cells after 48 h of etoposide treatment was analyzed by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells are the DIOC−, PI− and size− cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control mock, except where indicated ( n =3; n.s.: P > 0.05; *: P ⩽0.05; ***: P ⩽0.001). ( c,d ) PUMA, cleaved caspase-3 and PARP (full-length and cleaved forms) levels were analyzed by western blot in FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or mock stably transfected SH-SY5Y cells after 0, 4, 8 or 16 h of etoposide treatment. Actin detection was used as control
Figure Legend Snippet: FGF1 phosphorylation inhibits its anti-apoptotic activity in SH-SY5Y cells. SH-SY5Y cells were stably transfected with FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or empty (mock) expression vectors. ( a ) FGF1 levels were examined by western blot after heparin-sepharose concentration in cell lysates and conditioned media from SH-SY5Y cells expressing FGF1 WT , FGF1 S130A , FGF1 S130D or transfected with an empty vector (mock). ( b ) Apoptosis in FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or mock stably transfected SH-SY5Y cells after 48 h of etoposide treatment was analyzed by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells are the DIOC−, PI− and size− cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control mock, except where indicated ( n =3; n.s.: P > 0.05; *: P ⩽0.05; ***: P ⩽0.001). ( c,d ) PUMA, cleaved caspase-3 and PARP (full-length and cleaved forms) levels were analyzed by western blot in FGF1 WT , FGF1 K132E , FGF1 S130A , FGF S130D or mock stably transfected SH-SY5Y cells after 0, 4, 8 or 16 h of etoposide treatment. Actin detection was used as control

Techniques Used: Activity Assay, Stable Transfection, Transfection, Expressing, Western Blot, Concentration Assay, Plasmid Preparation, Flow Cytometry, Cytometry, Staining

7) Product Images from "Contributions of the D-Ring to the Activity of Etoposide Against Human Topoisomerase II?: Potential Interactions with DNA in the Ternary Enzyme-Drug-DNA Complex †"

Article Title: Contributions of the D-Ring to the Activity of Etoposide Against Human Topoisomerase II?: Potential Interactions with DNA in the Ternary Enzyme-Drug-DNA Complex †

Journal: Biochemistry

doi: 10.1021/bi200531q

Oligonucleotide sequence specificity of etoposide (closed circles) versus retroetoposide (open circles). Drug titrations were performed using an oligonucleotide substrate that contained a strong cleavage site for human topoisomerase IIα. The oligonucleotide was synthesized with either a C, A, T, or G at the −1 position relative to the scissile bond. The inset in the lower right panel compares DNA cleavage levels induced by 250 μM etoposide (closed bars) or retroetoposide (open bars) relative to cleavage of the oligonucleotide containing a C at the −1 position, which was set at 1 for both drugs. Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Oligonucleotide sequence specificity of etoposide (closed circles) versus retroetoposide (open circles). Drug titrations were performed using an oligonucleotide substrate that contained a strong cleavage site for human topoisomerase IIα. The oligonucleotide was synthesized with either a C, A, T, or G at the −1 position relative to the scissile bond. The inset in the lower right panel compares DNA cleavage levels induced by 250 μM etoposide (closed bars) or retroetoposide (open bars) relative to cleavage of the oligonucleotide containing a C at the −1 position, which was set at 1 for both drugs. Error bars represent the standard deviation of three independent experiments.

Techniques Used: Sequencing, Synthesized, Standard Deviation

DNA cleavage site specificity and utilization by human topoisomerase IIα in the presence of etoposide derivatives. A singly end-labeled linear 4332 bp fragment of pBR322 was used as the cleavage substrate. An autoradiogram of a polyacrylamide gel is shown. DNA cleavage reactions contained topoisomerase IIα with no drug (Topo), 10 μM etoposide (Etop), 250 μM retroetoposide (rEtop), 25 μM DEPT (DEPT), 250 μM retroDEPT (rDEPT), or 250 μM D-ring diol (DRD). A DNA control (DNA) also is shown. Data are representative of four independent experiments.
Figure Legend Snippet: DNA cleavage site specificity and utilization by human topoisomerase IIα in the presence of etoposide derivatives. A singly end-labeled linear 4332 bp fragment of pBR322 was used as the cleavage substrate. An autoradiogram of a polyacrylamide gel is shown. DNA cleavage reactions contained topoisomerase IIα with no drug (Topo), 10 μM etoposide (Etop), 250 μM retroetoposide (rEtop), 25 μM DEPT (DEPT), 250 μM retroDEPT (rDEPT), or 250 μM D-ring diol (DRD). A DNA control (DNA) also is shown. Data are representative of four independent experiments.

Techniques Used: Labeling

Summary of etoposide substituents that interact with human topoisomerase IIα. Protons that interact with the enzyme (as determined by STD 1 ). We propose that interactions between etoposide and DNA in the ternary complex (shaded in gray) are driven primarily by the D-ring, with additional contributions from the C4 sugar.
Figure Legend Snippet: Summary of etoposide substituents that interact with human topoisomerase IIα. Protons that interact with the enzyme (as determined by STD 1 ). We propose that interactions between etoposide and DNA in the ternary complex (shaded in gray) are driven primarily by the D-ring, with additional contributions from the C4 sugar.

Techniques Used:

Effects of etoposide derivatives on DNA cleavage mediated by human topoisomerase IIα. Levels of double-stranded DNA cleavage were expressed as a fold–enhancement over reactions that were carried out in the absence of drug. Assay mixtures contained 0–200 μM etoposide (closed circles), DEPT (closed squares), retroetoposide (rEtop, open circles), retroDEPT (rDEPT, open squares), or D-ring diol (open triangles). Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Effects of etoposide derivatives on DNA cleavage mediated by human topoisomerase IIα. Levels of double-stranded DNA cleavage were expressed as a fold–enhancement over reactions that were carried out in the absence of drug. Assay mixtures contained 0–200 μM etoposide (closed circles), DEPT (closed squares), retroetoposide (rEtop, open circles), retroDEPT (rDEPT, open squares), or D-ring diol (open triangles). Error bars represent the standard deviation of three independent experiments.

Techniques Used: Standard Deviation

Left: DNA ligation was examined in the absence of compound (no drug, closed triangles) or in the presence of 100 μM etoposide (closed circles), DEPT (closed squares), retroetoposide (rEtop, open circles), retroDEPT (rDEPT, open squares), or D-ring diol (open triangles). Right: Comparison of DNA cleavage (closed bars, 100 μM drug) and ligation (open bars, 30 s) mediated by human topoisomerase IIα in the presence of etoposide (E), DEPT (D), retroetoposide (rE), retroDEPT (rD), or D-ring diol (DRD). Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Left: DNA ligation was examined in the absence of compound (no drug, closed triangles) or in the presence of 100 μM etoposide (closed circles), DEPT (closed squares), retroetoposide (rEtop, open circles), retroDEPT (rDEPT, open squares), or D-ring diol (open triangles). Right: Comparison of DNA cleavage (closed bars, 100 μM drug) and ligation (open bars, 30 s) mediated by human topoisomerase IIα in the presence of etoposide (E), DEPT (D), retroetoposide (rE), retroDEPT (rD), or D-ring diol (DRD). Error bars represent the standard deviation of three independent experiments.

Techniques Used: DNA Ligation, Ligation, Standard Deviation

Levels of topoisomerase IIα-DNA cleavage complexes formed in human CEM leukemia cells that were treated with etoposide derivatives. DNA samples (10 μg) from cultures treated with no drug (ND) or 25 μM etoposide (E), DEPT (D), retroetoposide (rE), retroDEPT (rD), or D-ring diol (DRD) for 1 h were blotted onto a nitrocellulose membrane and probed with a polyclonal antibody directed against human topoisomerase IIα. Error bars represent the standard deviation of three independent experiments. A representative blot is shown above.
Figure Legend Snippet: Levels of topoisomerase IIα-DNA cleavage complexes formed in human CEM leukemia cells that were treated with etoposide derivatives. DNA samples (10 μg) from cultures treated with no drug (ND) or 25 μM etoposide (E), DEPT (D), retroetoposide (rE), retroDEPT (rD), or D-ring diol (DRD) for 1 h were blotted onto a nitrocellulose membrane and probed with a polyclonal antibody directed against human topoisomerase IIα. Error bars represent the standard deviation of three independent experiments. A representative blot is shown above.

Techniques Used: Standard Deviation

Competition between the D-ring diol and etoposide in the ternary complex. Topoisomerase IIα-mediated DNA cleavage in the presence of 50 μM etoposide and increasing concentrations (up to 500 μM) of D-ring diol is shown as fold–enhancement over etoposide alone. Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Competition between the D-ring diol and etoposide in the ternary complex. Topoisomerase IIα-mediated DNA cleavage in the presence of 50 μM etoposide and increasing concentrations (up to 500 μM) of D-ring diol is shown as fold–enhancement over etoposide alone. Error bars represent the standard deviation of three independent experiments.

Techniques Used: Standard Deviation

Structures of etoposide, retroetoposide, DEPT, retroDEPT and the D-ring diol.
Figure Legend Snippet: Structures of etoposide, retroetoposide, DEPT, retroDEPT and the D-ring diol.

Techniques Used:

8) Product Images from "Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types"

Article Title: Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047519

Effect of BH3-only proteins silencing on the etoposide-induced cell death . HepG2 cells were transfected with 50 nM BIM (A, B), NOXA (C, D) or BAD (E, F) siRNAs, or 25 nM BIM combined with 25 nM NOXA (G, H), or 50 nM RISC-free (RF) control siRNA or left untransfected (CTL) for 24 hours. 6 hours later (or 30 hours later for A and B), cells were incubated under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) with (ETO) or without (CTL) etoposide (50 µM) for 16 (A, C, E, G) or 40 hours (B, D, F, H). (A, C, E, G) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescence units (RFU) as means ±1 SD (n = 3). (B, D, F, H) LDH release was assessed. Results are presented in percentages as means ±1 SD (n = 4, but n = 3 in B for the condition HE CTL and in f for the condition H BAD). (A-H) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P
Figure Legend Snippet: Effect of BH3-only proteins silencing on the etoposide-induced cell death . HepG2 cells were transfected with 50 nM BIM (A, B), NOXA (C, D) or BAD (E, F) siRNAs, or 25 nM BIM combined with 25 nM NOXA (G, H), or 50 nM RISC-free (RF) control siRNA or left untransfected (CTL) for 24 hours. 6 hours later (or 30 hours later for A and B), cells were incubated under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) with (ETO) or without (CTL) etoposide (50 µM) for 16 (A, C, E, G) or 40 hours (B, D, F, H). (A, C, E, G) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescence units (RFU) as means ±1 SD (n = 3). (B, D, F, H) LDH release was assessed. Results are presented in percentages as means ±1 SD (n = 4, but n = 3 in B for the condition HE CTL and in f for the condition H BAD). (A-H) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P

Techniques Used: Transfection, CTL Assay, Incubation, Activity Assay, Fluorescence

Effect of p53 silencing on the expression of BIM, NOXA and BAD. HepG2 cells were transfected with 50 nM p53 siRNA (p53) or non-targeting control siRNA (NT) or left untransfected (/) for 24 hours. Minimum 6 hours later, cells were incubated under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) with (ETO) or without (CTL) etoposide (50 µM) for 16 hours. Proteins were detected in total cell extracts by western blotting, using specific antibodies. ß-actin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 .
Figure Legend Snippet: Effect of p53 silencing on the expression of BIM, NOXA and BAD. HepG2 cells were transfected with 50 nM p53 siRNA (p53) or non-targeting control siRNA (NT) or left untransfected (/) for 24 hours. Minimum 6 hours later, cells were incubated under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) with (ETO) or without (CTL) etoposide (50 µM) for 16 hours. Proteins were detected in total cell extracts by western blotting, using specific antibodies. ß-actin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 .

Techniques Used: Expressing, Transfection, Incubation, CTL Assay, Western Blot

Effects of hypoxia on drug-induced damage. (A) Effect of hypoxia, etoposide and paclitaxel on the abundance and phosphorylation of ATM. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 1 hour under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells. ATM and P-ATM were detected in nuclear protein extracts by western blotting using specific antibodies. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) Effect of hypoxia, etoposide and paclitaxel on microtubules. HepG2 cells were incubated 16 hours under normoxia (21% O 2 ) or hypoxia (1% O 2 ) in the presence or not of etoposide (50 µM) or paclitaxel (10 µM). After the incubation, cells were fixed, permeabilised and stained for alpha-tubulin using a specific antibody. Observation was performed using a confocal microscope with a constant photomultiplier.
Figure Legend Snippet: Effects of hypoxia on drug-induced damage. (A) Effect of hypoxia, etoposide and paclitaxel on the abundance and phosphorylation of ATM. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 1 hour under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells. ATM and P-ATM were detected in nuclear protein extracts by western blotting using specific antibodies. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) Effect of hypoxia, etoposide and paclitaxel on microtubules. HepG2 cells were incubated 16 hours under normoxia (21% O 2 ) or hypoxia (1% O 2 ) in the presence or not of etoposide (50 µM) or paclitaxel (10 µM). After the incubation, cells were fixed, permeabilised and stained for alpha-tubulin using a specific antibody. Observation was performed using a confocal microscope with a constant photomultiplier.

Techniques Used: Multiple Displacement Amplification, Incubation, CTL Assay, Western Blot, Staining, Microscopy

Effect of hypoxia, etoposide and paclitaxel on HIF-1alpha abundance and HIF-1 activity. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) (A-D) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells (A, C, D). (A) HIF-1alpha was detected in total cell extracts by western blotting using a specific antibody. Alpha-tubulin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) Before incubation, cells were co-transfected with the pGL3-(PGK-HRE6)-tk-luc reporter plasmid encoding the firefly luciferase and the pCMVß normalisation plasmid. Results are expressed as mean of the ratio between firefly luciferase activity and ß-galactosidase activity ±1 SD (n = 3). Statistical analysis was carried out with ANOVA 1. ***: P
Figure Legend Snippet: Effect of hypoxia, etoposide and paclitaxel on HIF-1alpha abundance and HIF-1 activity. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) (A-D) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells (A, C, D). (A) HIF-1alpha was detected in total cell extracts by western blotting using a specific antibody. Alpha-tubulin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) Before incubation, cells were co-transfected with the pGL3-(PGK-HRE6)-tk-luc reporter plasmid encoding the firefly luciferase and the pCMVß normalisation plasmid. Results are expressed as mean of the ratio between firefly luciferase activity and ß-galactosidase activity ±1 SD (n = 3). Statistical analysis was carried out with ANOVA 1. ***: P

Techniques Used: Activity Assay, Multiple Displacement Amplification, Incubation, CTL Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase

Effect of hypoxia on chemotherapeutic agents-induced cell death. HepG2, A549, U2OS, MDA-MB231, HT-29, Hep3B and PC-3 cells were incubated under normoxia (21% O 2 ) or hypoxia (1% O 2 ) in the absence (CTL) or presence of etoposide (ETO), paclitaxel (TAX), cisplatin (CIS) or camptothecin (CPT) for 16 (A, C, E, G, J) or 40 hours (B, D, F, H, I, K). The concentration used for each molecule is indicated in the graphs (in µM). (A, C, E, G, J) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescent units (RFU) as means ±1 SD (n = 3). (B, D, F, H, I, K) LDH release was assessed. Results are presented in percentages as means ±1 SD (n = 3). (A-K) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P
Figure Legend Snippet: Effect of hypoxia on chemotherapeutic agents-induced cell death. HepG2, A549, U2OS, MDA-MB231, HT-29, Hep3B and PC-3 cells were incubated under normoxia (21% O 2 ) or hypoxia (1% O 2 ) in the absence (CTL) or presence of etoposide (ETO), paclitaxel (TAX), cisplatin (CIS) or camptothecin (CPT) for 16 (A, C, E, G, J) or 40 hours (B, D, F, H, I, K). The concentration used for each molecule is indicated in the graphs (in µM). (A, C, E, G, J) Caspase-3/7 activity was assayed by measuring the fluorescence of free AFC released from the cleavage of the caspase-3/7 specific substrate Ac-DEVD-AFC. Results are expressed in relative fluorescent units (RFU) as means ±1 SD (n = 3). (B, D, F, H, I, K) LDH release was assessed. Results are presented in percentages as means ±1 SD (n = 3). (A-K) Statistical analysis was carried out with ANOVA 1. ns: non-significant; *: P

Techniques Used: Multiple Displacement Amplification, Incubation, CTL Assay, Cycling Probe Technology, Concentration Assay, Activity Assay, Fluorescence

Effect of hypoxia, etoposide and paclitaxel on the abundance and localisation of BCL-2 family proteins. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells. (A) Proteins were detected in total cell extracts by western blotting, using specific antibodies. alpha-tubulin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) After the incubation, subcellular fractionation was performed and proteins were detected in the MLP (mitochondria-lysosome-peroxisome) and S (cytosolic) fractions by western blotting, using specific antibodies. TOM20 and β-actin were used as loading controls for the MLP and S fractions respectively. One experiment representative out of three. Uncropped western blots are presented in Figure S1 .
Figure Legend Snippet: Effect of hypoxia, etoposide and paclitaxel on the abundance and localisation of BCL-2 family proteins. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not (CTL) of etoposide (ETO, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (TAX, 10 µM) in HepG2 cells. (A) Proteins were detected in total cell extracts by western blotting, using specific antibodies. alpha-tubulin was used as loading control. One experiment representative out of three. Uncropped western blots are presented in Figure S1 . (B) After the incubation, subcellular fractionation was performed and proteins were detected in the MLP (mitochondria-lysosome-peroxisome) and S (cytosolic) fractions by western blotting, using specific antibodies. TOM20 and β-actin were used as loading controls for the MLP and S fractions respectively. One experiment representative out of three. Uncropped western blots are presented in Figure S1 .

Techniques Used: Multiple Displacement Amplification, Incubation, CTL Assay, Western Blot, Fractionation

Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway. Results were obtained using “TLDA Human Apoptosis Panel” (Applied Biosystems) (TLDA). HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to TLDA analysis. 18S was used as housekeeping gene for data normalization. Actual numerical values are provided in Table S2 . Genes shown in bold are genes whose expression was validated by qRT-PCT.
Figure Legend Snippet: Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway. Results were obtained using “TLDA Human Apoptosis Panel” (Applied Biosystems) (TLDA). HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to TLDA analysis. 18S was used as housekeeping gene for data normalization. Actual numerical values are provided in Table S2 . Genes shown in bold are genes whose expression was validated by qRT-PCT.

Techniques Used: Expressing, TLDA Assay, Multiple Displacement Amplification, Incubation

Schematic representation of the effects of hypoxia on the etoposide-induced effects on BCL2-family proteins. (A) Schematic representation of the results obtained in figure 4 . Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red. The activation arrows and inhibition signs come from the hypothesis of the interactions between BCL-2 family proteins as explained in [69] , [70] . The proteins whose abundance is increased by etoposide are outlined with a thicker line. A yellow filling represents that, in the presence of etoposide, hypoxia decreased the abundance of the protein (or the death) as compared to normoxia. A pink filling represents that, in the presence of etoposide, hypoxia increased the abundance of the protein as compared to normoxia. (B) Schematic representation of the hypothetic relations between BIM, BAK, MCL-1 and NOXA. These possible mechanisms are represented for normoxic (21% O 2 ) or hypoxic (1% O 2 ) HepG2 cells incubated with or without etoposide (ETO; 50 µM) and with or without silencing (−) of NOXA and/or BIM. The hypothetic relations between these proteins are explained in details in the text. Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red.
Figure Legend Snippet: Schematic representation of the effects of hypoxia on the etoposide-induced effects on BCL2-family proteins. (A) Schematic representation of the results obtained in figure 4 . Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red. The activation arrows and inhibition signs come from the hypothesis of the interactions between BCL-2 family proteins as explained in [69] , [70] . The proteins whose abundance is increased by etoposide are outlined with a thicker line. A yellow filling represents that, in the presence of etoposide, hypoxia decreased the abundance of the protein (or the death) as compared to normoxia. A pink filling represents that, in the presence of etoposide, hypoxia increased the abundance of the protein as compared to normoxia. (B) Schematic representation of the hypothetic relations between BIM, BAK, MCL-1 and NOXA. These possible mechanisms are represented for normoxic (21% O 2 ) or hypoxic (1% O 2 ) HepG2 cells incubated with or without etoposide (ETO; 50 µM) and with or without silencing (−) of NOXA and/or BIM. The hypothetic relations between these proteins are explained in details in the text. Anti-apoptotic proteins are represented outlined in green and pro-apoptotic proteins are outlined in red.

Techniques Used: Activation Assay, Inhibition, Incubation

Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to real-time PCR in the presence of SYBR Green and specific primers. Actual numerical values are provided in Table S3 . Genes shown in bold are genes whose expression was assessed at the protein level by western blot analyses.
Figure Legend Snippet: Effect of hypoxia, etoposide and paclitaxel on the mRNA expression level of genes involved in the apoptotic pathway. HepG2, A549, MDA-MB231 and Hep3B cells were incubated 16 hours under normoxia (N, 21% O 2 ) or hypoxia (H, 1% O 2 ) in the presence or not of etoposide (E, 100 µM in Hep3B cells and 50 µM in the other cell types) or paclitaxel (T, 10 µM) in HepG2 cells. After incubation, total RNA was extracted, submitted to reverse transcription and then to real-time PCR in the presence of SYBR Green and specific primers. Actual numerical values are provided in Table S3 . Genes shown in bold are genes whose expression was assessed at the protein level by western blot analyses.

Techniques Used: Expressing, Multiple Displacement Amplification, Incubation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot

9) Product Images from "Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation"

Article Title: Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation

Journal: Scientific Reports

doi: 10.1038/s41598-018-32195-7

1p36 allelic loss is not observed in parental and HTLA-ER cells and miRNA-34a levels are reduced in both cell populations following etoposide treatment. ( A ) FISH analysis of HTLA-230 cells and HTLA-ER cells. Upper and lower left panels: metaphase of both HTLA-230 and HTLA-ER cells displays one normal chromosome 1 (close arrow) one 1 p arm derivative (close arrowhead) and 1 q arm derivative (open arrow); lower right panel: metaphase of HTLA-ER cells displaying one additional 1 p arm derivative (open arrowhead). ( B ) Expression levels of miRNA-34a in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: 1p36 allelic loss is not observed in parental and HTLA-ER cells and miRNA-34a levels are reduced in both cell populations following etoposide treatment. ( A ) FISH analysis of HTLA-230 cells and HTLA-ER cells. Upper and lower left panels: metaphase of both HTLA-230 and HTLA-ER cells displays one normal chromosome 1 (close arrow) one 1 p arm derivative (close arrowhead) and 1 q arm derivative (open arrow); lower right panel: metaphase of HTLA-ER cells displaying one additional 1 p arm derivative (open arrowhead). ( B ) Expression levels of miRNA-34a in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p

Techniques Used: Fluorescence In Situ Hybridization, Expressing

Parental and HTLA-ER cells express a non-inducible P53 protein carrying the homozygous TP53 missense mutation A161T. ( A ) Protein levels of P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. ( B ) Transactivation ability of wild-type and mutant (A161T) P53 proteins in yLFM-P21-5′, yLFM-BAX A + B and yLFM-MDM2P2C yeast strains. The transactivation ability was determined at two different temperatures (30 °C and 36 °C) using a constitutive expression of P53 proteins (ADH1 promoter). Presented data are the fold of induction over empty vector (pRS315) and standard deviation of four biological replicates. # p
Figure Legend Snippet: Parental and HTLA-ER cells express a non-inducible P53 protein carrying the homozygous TP53 missense mutation A161T. ( A ) Protein levels of P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. ( B ) Transactivation ability of wild-type and mutant (A161T) P53 proteins in yLFM-P21-5′, yLFM-BAX A + B and yLFM-MDM2P2C yeast strains. The transactivation ability was determined at two different temperatures (30 °C and 36 °C) using a constitutive expression of P53 proteins (ADH1 promoter). Presented data are the fold of induction over empty vector (pRS315) and standard deviation of four biological replicates. # p

Techniques Used: Mutagenesis, Western Blot, Expressing, Plasmid Preparation, Standard Deviation

Molecular mechanisms underlying the chemoresistance of HTLA-ER cells. This figure illustrates the observed molecular mechanisms underlying chemoresistance of HTLA-ER cells and the events leading to apoptosis in etoposide sensitive HTLA parental cells. Left panel: Short-term treatment with etoposide of HTLA-230 cells reduces oxidative phosphorylation and decreases glutathione (GSH) levels inducing reactive oxygen species (ROS) overproduction, thus leading to DNA damage (H2AX). Consequently, etoposide-induced genotoxic stress increases pro-apoptotic Bax, reduces anti-apoptotic Bcl2 and stimulates P53-Ser15 phosphorylation, two events leading to apoptosis and chemosensitivity. Right panel: HTLA-ER cells are able to efficiently counteract etoposide-induced ROS production by maintaining an efficient aerobic metabolism and increasing GSH levels. Long-term treatment with etoposide causes a deletion of the 13q14.3 locus and the consequent downregulation of miRNAs 15a/16-1, stimulating several pro-survival signals which contribute to inducing chemoresistance.
Figure Legend Snippet: Molecular mechanisms underlying the chemoresistance of HTLA-ER cells. This figure illustrates the observed molecular mechanisms underlying chemoresistance of HTLA-ER cells and the events leading to apoptosis in etoposide sensitive HTLA parental cells. Left panel: Short-term treatment with etoposide of HTLA-230 cells reduces oxidative phosphorylation and decreases glutathione (GSH) levels inducing reactive oxygen species (ROS) overproduction, thus leading to DNA damage (H2AX). Consequently, etoposide-induced genotoxic stress increases pro-apoptotic Bax, reduces anti-apoptotic Bcl2 and stimulates P53-Ser15 phosphorylation, two events leading to apoptosis and chemosensitivity. Right panel: HTLA-ER cells are able to efficiently counteract etoposide-induced ROS production by maintaining an efficient aerobic metabolism and increasing GSH levels. Long-term treatment with etoposide causes a deletion of the 13q14.3 locus and the consequent downregulation of miRNAs 15a/16-1, stimulating several pro-survival signals which contribute to inducing chemoresistance.

Techniques Used:

P53 Ser15 phosphorylation is detected only in etoposide-treated HTLA parental cells and is associated with PPM1D up-regulation. ( A ) Protein levels of phospho-(Ser15)-P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of phospho-P53/P53 ratio means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: P53 Ser15 phosphorylation is detected only in etoposide-treated HTLA parental cells and is associated with PPM1D up-regulation. ( A ) Protein levels of phospho-(Ser15)-P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of phospho-P53/P53 ratio means ± S.E.M of three independent experiments. ** p

Techniques Used: Western Blot

HTLA-ER cells have a deletion at the 13q14.3 locus which is associated with decreased levels of miRNAs 15a/16-1 in respect to parental cells. ( A ) FISH analysis of HTLA-230 and HTLA-ER cells: Upper panels: nuclei of HTLA-230 cells with two CEP12, two 13q34 and two D13S319 signals; nuclei of HTLA-ER cells with two CEP12, three 13q34 and one D13S319 signals. Lower panels: metaphase of HTLA-230 cells with two chromosomes 13 displaying 13q34 and D13S319 signals; metaphase of HTLA-ER cells with one chromosome 13 displaying 13q34 and D13S319 signals, one rearranged chromosome 13 displaying two 13q34, and one chromosome 12 displaying cep 12signal. ( B ) Expression levels of miRNA-15a (left panel) and miRNA-16 (right panel) in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: HTLA-ER cells have a deletion at the 13q14.3 locus which is associated with decreased levels of miRNAs 15a/16-1 in respect to parental cells. ( A ) FISH analysis of HTLA-230 and HTLA-ER cells: Upper panels: nuclei of HTLA-230 cells with two CEP12, two 13q34 and two D13S319 signals; nuclei of HTLA-ER cells with two CEP12, three 13q34 and one D13S319 signals. Lower panels: metaphase of HTLA-230 cells with two chromosomes 13 displaying 13q34 and D13S319 signals; metaphase of HTLA-ER cells with one chromosome 13 displaying 13q34 and D13S319 signals, one rearranged chromosome 13 displaying two 13q34, and one chromosome 12 displaying cep 12signal. ( B ) Expression levels of miRNA-15a (left panel) and miRNA-16 (right panel) in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p

Techniques Used: Fluorescence In Situ Hybridization, Expressing

The mitotic index of HTLA-ER cells and their Bax/Bcl2 ratio were not modified by acute etoposide exposure. ( A ) Mitotic index of HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.D. of four independent experiments per experimental condition (at least 4 × 10 3 cells per experimental condition were counted) ** p
Figure Legend Snippet: The mitotic index of HTLA-ER cells and their Bax/Bcl2 ratio were not modified by acute etoposide exposure. ( A ) Mitotic index of HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.D. of four independent experiments per experimental condition (at least 4 × 10 3 cells per experimental condition were counted) ** p

Techniques Used: Modification

BMI-1 overexpression with consequent p16 down-regulation is found in HTLA-ER cells in respect to parental cells. ( A ) Protein levels of BMI-1 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of protein expression levels means, normalized to β-actin expression ± S.E.M of three independent experiments. * p
Figure Legend Snippet: BMI-1 overexpression with consequent p16 down-regulation is found in HTLA-ER cells in respect to parental cells. ( A ) Protein levels of BMI-1 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of protein expression levels means, normalized to β-actin expression ± S.E.M of three independent experiments. * p

Techniques Used: Over Expression, Western Blot, Expressing

10) Product Images from "Etoposide Quinone Is a Redox-Dependent Topoisomerase II Poison"

Article Title: Etoposide Quinone Is a Redox-Dependent Topoisomerase II Poison

Journal: Biochemistry

doi: 10.1021/bi200438m

Etoposide quinone rapidly inactivates human topoisomerase IIα. Enzyme was incubated in the absence (open circles, No Drug), or the presence of 30 µM etoposide quinone (closed circles, EQ), 30 µM etoposide quinone + DTT (closed
Figure Legend Snippet: Etoposide quinone rapidly inactivates human topoisomerase IIα. Enzyme was incubated in the absence (open circles, No Drug), or the presence of 30 µM etoposide quinone (closed circles, EQ), 30 µM etoposide quinone + DTT (closed

Techniques Used: Incubation

Etoposide quinone activity is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence of etoposide quinone without (TII+DNA) or with DTT (TII+DNA+DTT),
Figure Legend Snippet: Etoposide quinone activity is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence of etoposide quinone without (TII+DNA) or with DTT (TII+DNA+DTT),

Techniques Used: Activity Assay

Etoposide quinone does not require ATP for optimal topoisomerase IIα-mediated DNA cleavage. Topoisomerase IIα (TII) DNA cleavage reactions were performed with no drug, 30 µM etoposide (Etop), or 30 µM etoposide quinone
Figure Legend Snippet: Etoposide quinone does not require ATP for optimal topoisomerase IIα-mediated DNA cleavage. Topoisomerase IIα (TII) DNA cleavage reactions were performed with no drug, 30 µM etoposide (Etop), or 30 µM etoposide quinone

Techniques Used:

Etoposide quinone does not form DNA lesions that poison topoisomerase IIα. DNA was incubated (DNA Incubation) without (−) 30 µM etoposide quinone (EQ), or with (+) etoposide quinone in the absence or presence of DTT. DNA was purified
Figure Legend Snippet: Etoposide quinone does not form DNA lesions that poison topoisomerase IIα. DNA was incubated (DNA Incubation) without (−) 30 µM etoposide quinone (EQ), or with (+) etoposide quinone in the absence or presence of DTT. DNA was purified

Techniques Used: Incubation, Purification

Etoposide quinone enhances topoisomerase II-mediated DNA cleavage in the absence of a reducing agent. Plasmid DNA cleavage was monitored using agarose gel electrophoresis (top) and quantified (bottom). The positions of supercoiled (form I, FI), nicked
Figure Legend Snippet: Etoposide quinone enhances topoisomerase II-mediated DNA cleavage in the absence of a reducing agent. Plasmid DNA cleavage was monitored using agarose gel electrophoresis (top) and quantified (bottom). The positions of supercoiled (form I, FI), nicked

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

Etoposide quinone induces higher levels of DNA cleavage than etoposide in the absence of DTT. Left panel , plasmid DNA cleavage reactions were performed in the presence of increasing concentrations of etoposide or etoposide quinone with or without DTT.
Figure Legend Snippet: Etoposide quinone induces higher levels of DNA cleavage than etoposide in the absence of DTT. Left panel , plasmid DNA cleavage reactions were performed in the presence of increasing concentrations of etoposide or etoposide quinone with or without DTT.

Techniques Used: Plasmid Preparation

Etoposide quinone inhibits topoisomerase II-mediated DNA ligation. DNA cleavage reactions were initiated in the absence (open triangles, No Drug) or presence (closed circles, EQ) of 30 µM etoposide quinone, 30 µM etoposide quinone + DTT
Figure Legend Snippet: Etoposide quinone inhibits topoisomerase II-mediated DNA ligation. DNA cleavage reactions were initiated in the absence (open triangles, No Drug) or presence (closed circles, EQ) of 30 µM etoposide quinone, 30 µM etoposide quinone + DTT

Techniques Used: DNA Ligation

Etoposide quinone induces a high ratio of double-stranded to single-stranded DNA breaks. DNA strand breaks were monitored in reactions with no drug (No Drug), 30 µM etoposide (Etop), 30 µM etoposide quinone (EQ), or 25 µM 1,4-benzoquinone
Figure Legend Snippet: Etoposide quinone induces a high ratio of double-stranded to single-stranded DNA breaks. DNA strand breaks were monitored in reactions with no drug (No Drug), 30 µM etoposide (Etop), 30 µM etoposide quinone (EQ), or 25 µM 1,4-benzoquinone

Techniques Used:

Metabolism of etoposide. The metabolism of etoposide can involve hydrolysis, sulfate modification, and demethylation (CYP3A4). While hydrolysis of etoposide yields hydroxy acid, the major metabolite, CYP3A4 metabolizes etoposide to the catechol. The parent
Figure Legend Snippet: Metabolism of etoposide. The metabolism of etoposide can involve hydrolysis, sulfate modification, and demethylation (CYP3A4). While hydrolysis of etoposide yields hydroxy acid, the major metabolite, CYP3A4 metabolizes etoposide to the catechol. The parent

Techniques Used: Modification

11) Product Images from "Etoposide Quinone Is a Redox-Dependent Topoisomerase II Poison"

Article Title: Etoposide Quinone Is a Redox-Dependent Topoisomerase II Poison

Journal: Biochemistry

doi: 10.1021/bi200438m

Etoposide quinone rapidly inactivates human topoisomerase IIα. Enzyme was incubated in the absence (open circles, No Drug), or the presence of 30 µM etoposide quinone (closed circles, EQ), 30 µM etoposide quinone + DTT (closed
Figure Legend Snippet: Etoposide quinone rapidly inactivates human topoisomerase IIα. Enzyme was incubated in the absence (open circles, No Drug), or the presence of 30 µM etoposide quinone (closed circles, EQ), 30 µM etoposide quinone + DTT (closed

Techniques Used: Incubation

Etoposide quinone activity is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence of etoposide quinone without (TII+DNA) or with DTT (TII+DNA+DTT),
Figure Legend Snippet: Etoposide quinone activity is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence of etoposide quinone without (TII+DNA) or with DTT (TII+DNA+DTT),

Techniques Used: Activity Assay

Etoposide quinone does not require ATP for optimal topoisomerase IIα-mediated DNA cleavage. Topoisomerase IIα (TII) DNA cleavage reactions were performed with no drug, 30 µM etoposide (Etop), or 30 µM etoposide quinone
Figure Legend Snippet: Etoposide quinone does not require ATP for optimal topoisomerase IIα-mediated DNA cleavage. Topoisomerase IIα (TII) DNA cleavage reactions were performed with no drug, 30 µM etoposide (Etop), or 30 µM etoposide quinone

Techniques Used:

Etoposide quinone does not form DNA lesions that poison topoisomerase IIα. DNA was incubated (DNA Incubation) without (−) 30 µM etoposide quinone (EQ), or with (+) etoposide quinone in the absence or presence of DTT. DNA was purified
Figure Legend Snippet: Etoposide quinone does not form DNA lesions that poison topoisomerase IIα. DNA was incubated (DNA Incubation) without (−) 30 µM etoposide quinone (EQ), or with (+) etoposide quinone in the absence or presence of DTT. DNA was purified

Techniques Used: Incubation, Purification

Etoposide quinone enhances topoisomerase II-mediated DNA cleavage in the absence of a reducing agent. Plasmid DNA cleavage was monitored using agarose gel electrophoresis (top) and quantified (bottom). The positions of supercoiled (form I, FI), nicked
Figure Legend Snippet: Etoposide quinone enhances topoisomerase II-mediated DNA cleavage in the absence of a reducing agent. Plasmid DNA cleavage was monitored using agarose gel electrophoresis (top) and quantified (bottom). The positions of supercoiled (form I, FI), nicked

Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

Etoposide quinone induces higher levels of DNA cleavage than etoposide in the absence of DTT. Left panel , plasmid DNA cleavage reactions were performed in the presence of increasing concentrations of etoposide or etoposide quinone with or without DTT.
Figure Legend Snippet: Etoposide quinone induces higher levels of DNA cleavage than etoposide in the absence of DTT. Left panel , plasmid DNA cleavage reactions were performed in the presence of increasing concentrations of etoposide or etoposide quinone with or without DTT.

Techniques Used: Plasmid Preparation

Etoposide quinone inhibits topoisomerase II-mediated DNA ligation. DNA cleavage reactions were initiated in the absence (open triangles, No Drug) or presence (closed circles, EQ) of 30 µM etoposide quinone, 30 µM etoposide quinone + DTT
Figure Legend Snippet: Etoposide quinone inhibits topoisomerase II-mediated DNA ligation. DNA cleavage reactions were initiated in the absence (open triangles, No Drug) or presence (closed circles, EQ) of 30 µM etoposide quinone, 30 µM etoposide quinone + DTT

Techniques Used: DNA Ligation

Etoposide quinone induces a high ratio of double-stranded to single-stranded DNA breaks. DNA strand breaks were monitored in reactions with no drug (No Drug), 30 µM etoposide (Etop), 30 µM etoposide quinone (EQ), or 25 µM 1,4-benzoquinone
Figure Legend Snippet: Etoposide quinone induces a high ratio of double-stranded to single-stranded DNA breaks. DNA strand breaks were monitored in reactions with no drug (No Drug), 30 µM etoposide (Etop), 30 µM etoposide quinone (EQ), or 25 µM 1,4-benzoquinone

Techniques Used:

Metabolism of etoposide. The metabolism of etoposide can involve hydrolysis, sulfate modification, and demethylation (CYP3A4). While hydrolysis of etoposide yields hydroxy acid, the major metabolite, CYP3A4 metabolizes etoposide to the catechol. The parent
Figure Legend Snippet: Metabolism of etoposide. The metabolism of etoposide can involve hydrolysis, sulfate modification, and demethylation (CYP3A4). While hydrolysis of etoposide yields hydroxy acid, the major metabolite, CYP3A4 metabolizes etoposide to the catechol. The parent

Techniques Used: Modification

12) Product Images from "Numerical Analysis of Etoposide Induced DNA Breaks"

Article Title: Numerical Analysis of Etoposide Induced DNA Breaks

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005859

Time-dependent induction of DSBs and H2AX phosphorylation. Analysis of DSBs and H2AX phosphorylation in SV40-transformed fibroblasts treated with 3 nM CLM (a) or 250 µM etoposide (b) for 0, 20, 40, 80 or 160 minutes at 37°C before analysis of DSBs with neutral CFGE and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.
Figure Legend Snippet: Time-dependent induction of DSBs and H2AX phosphorylation. Analysis of DSBs and H2AX phosphorylation in SV40-transformed fibroblasts treated with 3 nM CLM (a) or 250 µM etoposide (b) for 0, 20, 40, 80 or 160 minutes at 37°C before analysis of DSBs with neutral CFGE and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.

Techniques Used: Transformation Assay

Strand breaks induced by etoposide and CLM. SV40-transformed fibroblasts treated with (a) CLM (0–30 nM) or (b), etoposide (0–450 µM) for 40 min at 37°C. The induced levels of TSBs and DSBs were measured with neutral and alkaline CFGE. As a control, we also treated cells with the SSB-inducer H 2 O 2 (200 µM) or DSB and SSB inducer CLM (15 nM) to demonstrate that neutral CFGE fails to detect SSBs (a, separate gel). Error bars represent variation in two separate experiments performed on two different days.
Figure Legend Snippet: Strand breaks induced by etoposide and CLM. SV40-transformed fibroblasts treated with (a) CLM (0–30 nM) or (b), etoposide (0–450 µM) for 40 min at 37°C. The induced levels of TSBs and DSBs were measured with neutral and alkaline CFGE. As a control, we also treated cells with the SSB-inducer H 2 O 2 (200 µM) or DSB and SSB inducer CLM (15 nM) to demonstrate that neutral CFGE fails to detect SSBs (a, separate gel). Error bars represent variation in two separate experiments performed on two different days.

Techniques Used: Transformation Assay

Effect on cell survival of etoposide- and CLM-induced DSBs and TSBs. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM for 40 minutes at 37°C before analysis of colony survival and levels of (a) TSBs or (b) DSBs by neutral and alkaline CFGEs calculated as described in materials and methods. Error bars represent variation in two separate experiments performed on two different days.
Figure Legend Snippet: Effect on cell survival of etoposide- and CLM-induced DSBs and TSBs. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM for 40 minutes at 37°C before analysis of colony survival and levels of (a) TSBs or (b) DSBs by neutral and alkaline CFGEs calculated as described in materials and methods. Error bars represent variation in two separate experiments performed on two different days.

Techniques Used: Transformation Assay

Etoposide-induced DNA breaks detected by CFGE. A homodimer of topoII binds and cleaves cellular DNA. Etoposide binds independently to each monomer to block religation and thereby locks the topoII-DNA complex. At low concentrations, only one of the topoII monomers will be bound by etoposide and unable to religate the break, resulting in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be generated (b). In CFGE cells are lysed and proteins removed from DNA by SDS and proteinase K, allowing detection of protein-linked SSBs and DSBs.
Figure Legend Snippet: Etoposide-induced DNA breaks detected by CFGE. A homodimer of topoII binds and cleaves cellular DNA. Etoposide binds independently to each monomer to block religation and thereby locks the topoII-DNA complex. At low concentrations, only one of the topoII monomers will be bound by etoposide and unable to religate the break, resulting in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be generated (b). In CFGE cells are lysed and proteins removed from DNA by SDS and proteinase K, allowing detection of protein-linked SSBs and DSBs.

Techniques Used: Blocking Assay, Generated

Cell survival and H2AX phosphorylation in response to etoposide or CLM. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of colony survival and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.
Figure Legend Snippet: Cell survival and H2AX phosphorylation in response to etoposide or CLM. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of colony survival and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.

Techniques Used: Transformation Assay

Induction of DSBs and H2AX phosphorylation at different cell-cycle stages. G361 cells were treated with 3 or 10 nM CLM (a) or 75 µM or 250 µM Etoposide (b) for 40 minutes before DNA staining, FACS sorting of G1, S and G2 cells, and analysis of DSBs by neutral CFGE. G361 cells were untreated or treated with 0.1 nM CLM or 75 µM Etoposide (c) for 40 minutes before DNA staining and analysis of H2AX phosphorylation and DNA-content to examine H2AX phosphorylation in G1, S and G2 cells. G361 cells were treated with CLM (d) or Etoposide (e) for 40 minutes before DNA staining and analysis of H2AX phosphorylation. Error bars represent variation from two separate experiments performed on two different days.
Figure Legend Snippet: Induction of DSBs and H2AX phosphorylation at different cell-cycle stages. G361 cells were treated with 3 or 10 nM CLM (a) or 75 µM or 250 µM Etoposide (b) for 40 minutes before DNA staining, FACS sorting of G1, S and G2 cells, and analysis of DSBs by neutral CFGE. G361 cells were untreated or treated with 0.1 nM CLM or 75 µM Etoposide (c) for 40 minutes before DNA staining and analysis of H2AX phosphorylation and DNA-content to examine H2AX phosphorylation in G1, S and G2 cells. G361 cells were treated with CLM (d) or Etoposide (e) for 40 minutes before DNA staining and analysis of H2AX phosphorylation. Error bars represent variation from two separate experiments performed on two different days.

Techniques Used: Staining, FACS

Induction of H2AX phosphorylation by etoposide- or CLM-induced DSBs. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of H2AX phosphorylation and DSB-level by neutral CFGE. Error bars represent variation in two separate experiments performed on different days.
Figure Legend Snippet: Induction of H2AX phosphorylation by etoposide- or CLM-induced DSBs. SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of H2AX phosphorylation and DSB-level by neutral CFGE. Error bars represent variation in two separate experiments performed on different days.

Techniques Used: Transformation Assay

Etoposide-induced DNA damage in cells. A homodimer of topoII binds and cleaves cellular DNA, generating a topoII-linked DSB. Etoposide binds independently to each monomer to block religation, locking the topoII monomer to the DNA break. If only one of the topoII monomers is bound by etoposide and unable to religate the break, this results in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be stabilized (b). TopoII-linked DNA breaks that are encountered by RNA or DNA polymerases during etoposide exposure will be denatured and therefore unable to religate the breaks. Denatured topoII will be cleared from the breaks, resulting in free DSBs that can induce H2AX phosphorylation. The relative amounts of these breaks as a percentage of all etoposide-induced breaks are indicated.
Figure Legend Snippet: Etoposide-induced DNA damage in cells. A homodimer of topoII binds and cleaves cellular DNA, generating a topoII-linked DSB. Etoposide binds independently to each monomer to block religation, locking the topoII monomer to the DNA break. If only one of the topoII monomers is bound by etoposide and unable to religate the break, this results in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be stabilized (b). TopoII-linked DNA breaks that are encountered by RNA or DNA polymerases during etoposide exposure will be denatured and therefore unable to religate the breaks. Denatured topoII will be cleared from the breaks, resulting in free DSBs that can induce H2AX phosphorylation. The relative amounts of these breaks as a percentage of all etoposide-induced breaks are indicated.

Techniques Used: Blocking Assay

13) Product Images from "FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization"

Article Title: FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization

Journal: Oncogenesis

doi: 10.1038/s41389-018-0033-y

p21 anti-apoptotic activities are not necessary for FGF1-induced resistance to etoposide. a COV434 Mock and COV434 FGF1 cells were transfected or not with scramble (scr) or p21 siRNA. Upper panel: average apoptosis rates ± SEM for 2 experiments done in duplicate were measured by flow cytometry in cells treated with etoposide for 6 h, or not treated (Ctl). Lower panel: Western blot analysis for p21 protein levels in COV434 Mock and COV434 FGF1 cells transfected with scr siRNA or p21 siRNA. b Western blot analysis of total proteins for p21 and for procaspase-9, cleaved caspase-9, and cleaved caspase-3, and PARP levels. COV434 Mock and FGF1 cells transfected with scr or p21 siRNA were treated or not with etoposide for 16 h. One experiment representative of 3 independent experiments is shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
Figure Legend Snippet: p21 anti-apoptotic activities are not necessary for FGF1-induced resistance to etoposide. a COV434 Mock and COV434 FGF1 cells were transfected or not with scramble (scr) or p21 siRNA. Upper panel: average apoptosis rates ± SEM for 2 experiments done in duplicate were measured by flow cytometry in cells treated with etoposide for 6 h, or not treated (Ctl). Lower panel: Western blot analysis for p21 protein levels in COV434 Mock and COV434 FGF1 cells transfected with scr siRNA or p21 siRNA. b Western blot analysis of total proteins for p21 and for procaspase-9, cleaved caspase-9, and cleaved caspase-3, and PARP levels. COV434 Mock and FGF1 cells transfected with scr or p21 siRNA were treated or not with etoposide for 16 h. One experiment representative of 3 independent experiments is shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Techniques Used: Transfection, Flow Cytometry, Cytometry, CTL Assay, Western Blot

p53 is involved in the induction of apoptosis in COV434 cells. a COV434 cells were transfected or not (NT) with scramble (scr) or p53 siRNA. Upper panel: histogram represents average apoptosis rates ± SEM for 2 experiments done in duplicate measured by flow cytometry in cells treated with etoposide for 17 h, or not treated (Ctl). Lower panel: Western blot analysis for p53 protein levels in COV434 cells transfected with scr siRNA or p53 siRNA. b COV434 cells were transfected with an empty vector (Mock), a vector encoding p53 wild-type (p53 WT ) and a vector encoding p53 fused to the mitochondrial transmembrane domain of BCL-X L (p53 CTB ). Cells were treated with of G418 for one week and then stained with ethidium bromide and visualized using Chemidoc (Biorad)
Figure Legend Snippet: p53 is involved in the induction of apoptosis in COV434 cells. a COV434 cells were transfected or not (NT) with scramble (scr) or p53 siRNA. Upper panel: histogram represents average apoptosis rates ± SEM for 2 experiments done in duplicate measured by flow cytometry in cells treated with etoposide for 17 h, or not treated (Ctl). Lower panel: Western blot analysis for p53 protein levels in COV434 cells transfected with scr siRNA or p53 siRNA. b COV434 cells were transfected with an empty vector (Mock), a vector encoding p53 wild-type (p53 WT ) and a vector encoding p53 fused to the mitochondrial transmembrane domain of BCL-X L (p53 CTB ). Cells were treated with of G418 for one week and then stained with ethidium bromide and visualized using Chemidoc (Biorad)

Techniques Used: Transfection, Flow Cytometry, Cytometry, CTL Assay, Western Blot, Plasmid Preparation, CtB Assay, Staining

FGF1 regulates p53-mitochondrial localization. a Average apoptosis rates ± SEM for 3 experiments done in triplicate were measured by flow cytometry in non-transfected (NT), mock and FGF1-overexpressing COV434 cells treated with etoposide for 16 h. These cells were pretreated or not with the p53 mitochondrial localization inhibitor pifithrin-mu (PFT-µ, 10 µM for 90 min). b Non-transfected COV434 cells were pretreated or not with PFT-µ (10 µM for 90 min) prior to etoposide treatment (25 µg/mL for 2h30). Mitochondrial localization of p53 was determined by western blot analysis of enriched mitochondrial fractions (upper panel). Quantification of mitochondrial p53 normalized to TOM40 for four experiments (lower panel). * Molecular weight lane. c Cytosolic, mitochondrial and total proteins of COV434-Mock, and COV434-FGF1 cells, treated or not with etoposide for 4 h, were analyzed for p53 and FGF1 localization by western blot (left panel). Quantification of mitochondrial p53 normalized to TOM40 (right panel). Results are from 6 independent experiments, means ± SEM, two-tailed t -test results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: FGF1 regulates p53-mitochondrial localization. a Average apoptosis rates ± SEM for 3 experiments done in triplicate were measured by flow cytometry in non-transfected (NT), mock and FGF1-overexpressing COV434 cells treated with etoposide for 16 h. These cells were pretreated or not with the p53 mitochondrial localization inhibitor pifithrin-mu (PFT-µ, 10 µM for 90 min). b Non-transfected COV434 cells were pretreated or not with PFT-µ (10 µM for 90 min) prior to etoposide treatment (25 µg/mL for 2h30). Mitochondrial localization of p53 was determined by western blot analysis of enriched mitochondrial fractions (upper panel). Quantification of mitochondrial p53 normalized to TOM40 for four experiments (lower panel). * Molecular weight lane. c Cytosolic, mitochondrial and total proteins of COV434-Mock, and COV434-FGF1 cells, treated or not with etoposide for 4 h, were analyzed for p53 and FGF1 localization by western blot (left panel). Quantification of mitochondrial p53 normalized to TOM40 (right panel). Results are from 6 independent experiments, means ± SEM, two-tailed t -test results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Flow Cytometry, Cytometry, Transfection, Western Blot, Molecular Weight, Two Tailed Test

FGF1 overexpression protects COV434 cells from etoposide-induced apoptosis. a Upper panel: Average flow cytometry quantification of apoptotic cells characterized by their low DIOC staining and cell condensation (DIOC - , Size - ) ± SEM for 3 experiments done in triplicate. Non-transfected COV434 (NT), two COV434-Mock clonal cell lines and three COV434-FGF1 clonal cell lines were treated with etoposide (25 µg/mL) for 16 h, or not treated (Ctl), The t-tests compare to NT Eto. Lower panel: FGF1 levels in non-transfected, mock and FGF1 overexpressing clones using western blot analysis. Total proteins are visualized with the Biorad stain free system. b Immunofluorescence study for cytochrome c release. COV434-Mock C1 and -FGF1 C1 cells were treated with 25 µg/mL etoposide for 4 h. Cells were stained with an anti-cytochrome c antibody (green) and TO-PRO-3 (blue) to visualize nuclei (left panels). Scale bar represents 40 µm. The histogram presents the average percentages ± SEM for 3 independent experiments of cells exhibiting cytochrome c release (right panel). The t-test compares to Mock cells similarly treated. c Upper panel: Western blot analysis of total proteins for procaspase-9, cleaved caspase-9 and −3 and PARP levels. COV434-Mock and -FGF1 cells were treated or not (0 h) with 25 µg/mL etoposide for 2, 4, 6, or 16 h. Lower panel: histograms present the average fold-change decrease of cleaved caspase-9, cleaved caspase-3 and cleaved PARP in COV434-FGF1 cells ± SEM from pooled results of three FGF1 overexpressing clones ( n = 8). Two-tailed unpaired t-tests results are shown as * for P ≤ 0.05, ** for P ≤ 0.01 and *** for P ≤ 0.001
Figure Legend Snippet: FGF1 overexpression protects COV434 cells from etoposide-induced apoptosis. a Upper panel: Average flow cytometry quantification of apoptotic cells characterized by their low DIOC staining and cell condensation (DIOC - , Size - ) ± SEM for 3 experiments done in triplicate. Non-transfected COV434 (NT), two COV434-Mock clonal cell lines and three COV434-FGF1 clonal cell lines were treated with etoposide (25 µg/mL) for 16 h, or not treated (Ctl), The t-tests compare to NT Eto. Lower panel: FGF1 levels in non-transfected, mock and FGF1 overexpressing clones using western blot analysis. Total proteins are visualized with the Biorad stain free system. b Immunofluorescence study for cytochrome c release. COV434-Mock C1 and -FGF1 C1 cells were treated with 25 µg/mL etoposide for 4 h. Cells were stained with an anti-cytochrome c antibody (green) and TO-PRO-3 (blue) to visualize nuclei (left panels). Scale bar represents 40 µm. The histogram presents the average percentages ± SEM for 3 independent experiments of cells exhibiting cytochrome c release (right panel). The t-test compares to Mock cells similarly treated. c Upper panel: Western blot analysis of total proteins for procaspase-9, cleaved caspase-9 and −3 and PARP levels. COV434-Mock and -FGF1 cells were treated or not (0 h) with 25 µg/mL etoposide for 2, 4, 6, or 16 h. Lower panel: histograms present the average fold-change decrease of cleaved caspase-9, cleaved caspase-3 and cleaved PARP in COV434-FGF1 cells ± SEM from pooled results of three FGF1 overexpressing clones ( n = 8). Two-tailed unpaired t-tests results are shown as * for P ≤ 0.05, ** for P ≤ 0.01 and *** for P ≤ 0.001

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Staining, Transfection, CTL Assay, Clone Assay, Western Blot, Immunofluorescence, Two Tailed Test

FGF1 overexpression attenuates the etoposide-induced G2/M cell cycle arrest. Non-transfected (NT), mock and FGF1-overexpressing COV434 cells were treated with etoposide for 16 h, or untreated (Ctl). DNA was stained with Hoechst 33342 and cellular content was analyzed by flow cytometry. a Cytograms showing cell cycle phase distribution (G1, S, G2/M). b The histograms show the average percentages of cells in each cell cycle phase ± SEM. Results for 4 independent experiments in replicate ( n = 10). T-tests compare to NT Eto. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
Figure Legend Snippet: FGF1 overexpression attenuates the etoposide-induced G2/M cell cycle arrest. Non-transfected (NT), mock and FGF1-overexpressing COV434 cells were treated with etoposide for 16 h, or untreated (Ctl). DNA was stained with Hoechst 33342 and cellular content was analyzed by flow cytometry. a Cytograms showing cell cycle phase distribution (G1, S, G2/M). b The histograms show the average percentages of cells in each cell cycle phase ± SEM. Results for 4 independent experiments in replicate ( n = 10). T-tests compare to NT Eto. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Techniques Used: Over Expression, Transfection, CTL Assay, Staining, Flow Cytometry, Cytometry

p53 transcriptional-dependent activities are dispensable for the apoptosis of the COV434 ovarian cell line. a COV434-Mock and COV434-FGF1 cells were treated or not with 25 µg/mL etoposide for 1, 2, 3 or 16 h. Total protein extracts were analyzed for p53 and Ser15-phosphorylated p53 by western blotting. b COV434 Mock and COV434-FGF1 cells were treated or not with etoposide for 1, 2, 3 or 16 h. Total proteins were analyzed for PUMA, Bax and p21 protein levels by western blotting. c Average apoptosis rates ± SEM for 2 experiments done in triplicate measured by flow cytometry of non-transfected COV434 (NT), Mock and FGF1 cells treated with etoposide for 16 h. Cells were pretreated or not for 90 min with the p53 transcriptional inhibitor pifithrin-alpha (PFT-α, 30 µM). d Western blot analysis of p21 and Bax protein levels in COV434 Mock and FGF1 cells pretreated with PFT-α (30 µM for 90 min), followed by an etoposide (25 µg/mL) treatment for 6 or 16 h. Two-tailed t -tests results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: p53 transcriptional-dependent activities are dispensable for the apoptosis of the COV434 ovarian cell line. a COV434-Mock and COV434-FGF1 cells were treated or not with 25 µg/mL etoposide for 1, 2, 3 or 16 h. Total protein extracts were analyzed for p53 and Ser15-phosphorylated p53 by western blotting. b COV434 Mock and COV434-FGF1 cells were treated or not with etoposide for 1, 2, 3 or 16 h. Total proteins were analyzed for PUMA, Bax and p21 protein levels by western blotting. c Average apoptosis rates ± SEM for 2 experiments done in triplicate measured by flow cytometry of non-transfected COV434 (NT), Mock and FGF1 cells treated with etoposide for 16 h. Cells were pretreated or not for 90 min with the p53 transcriptional inhibitor pifithrin-alpha (PFT-α, 30 µM). d Western blot analysis of p21 and Bax protein levels in COV434 Mock and FGF1 cells pretreated with PFT-α (30 µM for 90 min), followed by an etoposide (25 µg/mL) treatment for 6 or 16 h. Two-tailed t -tests results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Transfection, Two Tailed Test

Both FGFR-dependent and FGFR-independent pathways are involved in FGF1 anti-apoptotic activity. a Western blot analysis for FGF1 levels in total extracts and conditioned media of non-transfected COV434 (NT), COV434-Mock and COV434-FGF1 cells. Endogenous FGF1 is detected in all total extracts whereas exogenous FGF1-V5-His is seen only in FGF1-overexpressing cells as expected. b Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock cells. Cells were pretreated or not with the FGFR1/3 inhibitor PD173074 (25 nM for one hour), followed or not by a treatment with 50 ng/mL of recombinant FGF1 (rFGF1) supplemented with 10 µg/mL heparin for 24 h. On the next day, these treatments were renewed adding or not etoposide for 16 h. c Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock and COV434-FGF1 cells pretreated or not with 25 nM PD173074 for 24 h, and treated or not with etoposide (25 µg/mL for 16 h). Two-tailed t -tests are indicated by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: Both FGFR-dependent and FGFR-independent pathways are involved in FGF1 anti-apoptotic activity. a Western blot analysis for FGF1 levels in total extracts and conditioned media of non-transfected COV434 (NT), COV434-Mock and COV434-FGF1 cells. Endogenous FGF1 is detected in all total extracts whereas exogenous FGF1-V5-His is seen only in FGF1-overexpressing cells as expected. b Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock cells. Cells were pretreated or not with the FGFR1/3 inhibitor PD173074 (25 nM for one hour), followed or not by a treatment with 50 ng/mL of recombinant FGF1 (rFGF1) supplemented with 10 µg/mL heparin for 24 h. On the next day, these treatments were renewed adding or not etoposide for 16 h. c Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock and COV434-FGF1 cells pretreated or not with 25 nM PD173074 for 24 h, and treated or not with etoposide (25 µg/mL for 16 h). Two-tailed t -tests are indicated by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Activity Assay, Western Blot, Transfection, Flow Cytometry, Cytometry, Recombinant, Two Tailed Test

14) Product Images from "FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization"

Article Title: FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization

Journal: Oncogenesis

doi: 10.1038/s41389-018-0033-y

p21 anti-apoptotic activities are not necessary for FGF1-induced resistance to etoposide. a COV434 Mock and COV434 FGF1 cells were transfected or not with scramble (scr) or p21 siRNA. Upper panel: average apoptosis rates ± SEM for 2 experiments done in duplicate were measured by flow cytometry in cells treated with etoposide for 6 h, or not treated (Ctl). Lower panel: Western blot analysis for p21 protein levels in COV434 Mock and COV434 FGF1 cells transfected with scr siRNA or p21 siRNA. b Western blot analysis of total proteins for p21 and for procaspase-9, cleaved caspase-9, and cleaved caspase-3, and PARP levels. COV434 Mock and FGF1 cells transfected with scr or p21 siRNA were treated or not with etoposide for 16 h. One experiment representative of 3 independent experiments is shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
Figure Legend Snippet: p21 anti-apoptotic activities are not necessary for FGF1-induced resistance to etoposide. a COV434 Mock and COV434 FGF1 cells were transfected or not with scramble (scr) or p21 siRNA. Upper panel: average apoptosis rates ± SEM for 2 experiments done in duplicate were measured by flow cytometry in cells treated with etoposide for 6 h, or not treated (Ctl). Lower panel: Western blot analysis for p21 protein levels in COV434 Mock and COV434 FGF1 cells transfected with scr siRNA or p21 siRNA. b Western blot analysis of total proteins for p21 and for procaspase-9, cleaved caspase-9, and cleaved caspase-3, and PARP levels. COV434 Mock and FGF1 cells transfected with scr or p21 siRNA were treated or not with etoposide for 16 h. One experiment representative of 3 independent experiments is shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Techniques Used: Transfection, Flow Cytometry, Cytometry, CTL Assay, Western Blot

p53 is involved in the induction of apoptosis in COV434 cells. a COV434 cells were transfected or not (NT) with scramble (scr) or p53 siRNA. Upper panel: histogram represents average apoptosis rates ± SEM for 2 experiments done in duplicate measured by flow cytometry in cells treated with etoposide for 17 h, or not treated (Ctl). Lower panel: Western blot analysis for p53 protein levels in COV434 cells transfected with scr siRNA or p53 siRNA. b COV434 cells were transfected with an empty vector (Mock), a vector encoding p53 wild-type (p53 WT ) and a vector encoding p53 fused to the mitochondrial transmembrane domain of BCL-X L (p53 CTB ). Cells were treated with of G418 for one week and then stained with ethidium bromide and visualized using Chemidoc (Biorad)
Figure Legend Snippet: p53 is involved in the induction of apoptosis in COV434 cells. a COV434 cells were transfected or not (NT) with scramble (scr) or p53 siRNA. Upper panel: histogram represents average apoptosis rates ± SEM for 2 experiments done in duplicate measured by flow cytometry in cells treated with etoposide for 17 h, or not treated (Ctl). Lower panel: Western blot analysis for p53 protein levels in COV434 cells transfected with scr siRNA or p53 siRNA. b COV434 cells were transfected with an empty vector (Mock), a vector encoding p53 wild-type (p53 WT ) and a vector encoding p53 fused to the mitochondrial transmembrane domain of BCL-X L (p53 CTB ). Cells were treated with of G418 for one week and then stained with ethidium bromide and visualized using Chemidoc (Biorad)

Techniques Used: Transfection, Flow Cytometry, Cytometry, CTL Assay, Western Blot, Plasmid Preparation, CtB Assay, Staining

FGF1 regulates p53-mitochondrial localization. a Average apoptosis rates ± SEM for 3 experiments done in triplicate were measured by flow cytometry in non-transfected (NT), mock and FGF1-overexpressing COV434 cells treated with etoposide for 16 h. These cells were pretreated or not with the p53 mitochondrial localization inhibitor pifithrin-mu (PFT-µ, 10 µM for 90 min). b Non-transfected COV434 cells were pretreated or not with PFT-µ (10 µM for 90 min) prior to etoposide treatment (25 µg/mL for 2h30). Mitochondrial localization of p53 was determined by western blot analysis of enriched mitochondrial fractions (upper panel). Quantification of mitochondrial p53 normalized to TOM40 for four experiments (lower panel). * Molecular weight lane. c Cytosolic, mitochondrial and total proteins of COV434-Mock, and COV434-FGF1 cells, treated or not with etoposide for 4 h, were analyzed for p53 and FGF1 localization by western blot (left panel). Quantification of mitochondrial p53 normalized to TOM40 (right panel). Results are from 6 independent experiments, means ± SEM, two-tailed t -test results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: FGF1 regulates p53-mitochondrial localization. a Average apoptosis rates ± SEM for 3 experiments done in triplicate were measured by flow cytometry in non-transfected (NT), mock and FGF1-overexpressing COV434 cells treated with etoposide for 16 h. These cells were pretreated or not with the p53 mitochondrial localization inhibitor pifithrin-mu (PFT-µ, 10 µM for 90 min). b Non-transfected COV434 cells were pretreated or not with PFT-µ (10 µM for 90 min) prior to etoposide treatment (25 µg/mL for 2h30). Mitochondrial localization of p53 was determined by western blot analysis of enriched mitochondrial fractions (upper panel). Quantification of mitochondrial p53 normalized to TOM40 for four experiments (lower panel). * Molecular weight lane. c Cytosolic, mitochondrial and total proteins of COV434-Mock, and COV434-FGF1 cells, treated or not with etoposide for 4 h, were analyzed for p53 and FGF1 localization by western blot (left panel). Quantification of mitochondrial p53 normalized to TOM40 (right panel). Results are from 6 independent experiments, means ± SEM, two-tailed t -test results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Flow Cytometry, Cytometry, Transfection, Western Blot, Molecular Weight, Two Tailed Test

FGF1 overexpression protects COV434 cells from etoposide-induced apoptosis. a Upper panel: Average flow cytometry quantification of apoptotic cells characterized by their low DIOC staining and cell condensation (DIOC - , Size - ) ± SEM for 3 experiments done in triplicate. Non-transfected COV434 (NT), two COV434-Mock clonal cell lines and three COV434-FGF1 clonal cell lines were treated with etoposide (25 µg/mL) for 16 h, or not treated (Ctl), The t-tests compare to NT Eto. Lower panel: FGF1 levels in non-transfected, mock and FGF1 overexpressing clones using western blot analysis. Total proteins are visualized with the Biorad stain free system. b Immunofluorescence study for cytochrome c release. COV434-Mock C1 and -FGF1 C1 cells were treated with 25 µg/mL etoposide for 4 h. Cells were stained with an anti-cytochrome c antibody (green) and TO-PRO-3 (blue) to visualize nuclei (left panels). Scale bar represents 40 µm. The histogram presents the average percentages ± SEM for 3 independent experiments of cells exhibiting cytochrome c release (right panel). The t-test compares to Mock cells similarly treated. c Upper panel: Western blot analysis of total proteins for procaspase-9, cleaved caspase-9 and −3 and PARP levels. COV434-Mock and -FGF1 cells were treated or not (0 h) with 25 µg/mL etoposide for 2, 4, 6, or 16 h. Lower panel: histograms present the average fold-change decrease of cleaved caspase-9, cleaved caspase-3 and cleaved PARP in COV434-FGF1 cells ± SEM from pooled results of three FGF1 overexpressing clones ( n = 8). Two-tailed unpaired t-tests results are shown as * for P ≤ 0.05, ** for P ≤ 0.01 and *** for P ≤ 0.001
Figure Legend Snippet: FGF1 overexpression protects COV434 cells from etoposide-induced apoptosis. a Upper panel: Average flow cytometry quantification of apoptotic cells characterized by their low DIOC staining and cell condensation (DIOC - , Size - ) ± SEM for 3 experiments done in triplicate. Non-transfected COV434 (NT), two COV434-Mock clonal cell lines and three COV434-FGF1 clonal cell lines were treated with etoposide (25 µg/mL) for 16 h, or not treated (Ctl), The t-tests compare to NT Eto. Lower panel: FGF1 levels in non-transfected, mock and FGF1 overexpressing clones using western blot analysis. Total proteins are visualized with the Biorad stain free system. b Immunofluorescence study for cytochrome c release. COV434-Mock C1 and -FGF1 C1 cells were treated with 25 µg/mL etoposide for 4 h. Cells were stained with an anti-cytochrome c antibody (green) and TO-PRO-3 (blue) to visualize nuclei (left panels). Scale bar represents 40 µm. The histogram presents the average percentages ± SEM for 3 independent experiments of cells exhibiting cytochrome c release (right panel). The t-test compares to Mock cells similarly treated. c Upper panel: Western blot analysis of total proteins for procaspase-9, cleaved caspase-9 and −3 and PARP levels. COV434-Mock and -FGF1 cells were treated or not (0 h) with 25 µg/mL etoposide for 2, 4, 6, or 16 h. Lower panel: histograms present the average fold-change decrease of cleaved caspase-9, cleaved caspase-3 and cleaved PARP in COV434-FGF1 cells ± SEM from pooled results of three FGF1 overexpressing clones ( n = 8). Two-tailed unpaired t-tests results are shown as * for P ≤ 0.05, ** for P ≤ 0.01 and *** for P ≤ 0.001

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Staining, Transfection, CTL Assay, Clone Assay, Western Blot, Immunofluorescence, Two Tailed Test

FGF1 overexpression attenuates the etoposide-induced G2/M cell cycle arrest. Non-transfected (NT), mock and FGF1-overexpressing COV434 cells were treated with etoposide for 16 h, or untreated (Ctl). DNA was stained with Hoechst 33342 and cellular content was analyzed by flow cytometry. a Cytograms showing cell cycle phase distribution (G1, S, G2/M). b The histograms show the average percentages of cells in each cell cycle phase ± SEM. Results for 4 independent experiments in replicate ( n = 10). T-tests compare to NT Eto. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
Figure Legend Snippet: FGF1 overexpression attenuates the etoposide-induced G2/M cell cycle arrest. Non-transfected (NT), mock and FGF1-overexpressing COV434 cells were treated with etoposide for 16 h, or untreated (Ctl). DNA was stained with Hoechst 33342 and cellular content was analyzed by flow cytometry. a Cytograms showing cell cycle phase distribution (G1, S, G2/M). b The histograms show the average percentages of cells in each cell cycle phase ± SEM. Results for 4 independent experiments in replicate ( n = 10). T-tests compare to NT Eto. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Techniques Used: Over Expression, Transfection, CTL Assay, Staining, Flow Cytometry, Cytometry

p53 transcriptional-dependent activities are dispensable for the apoptosis of the COV434 ovarian cell line. a COV434-Mock and COV434-FGF1 cells were treated or not with 25 µg/mL etoposide for 1, 2, 3 or 16 h. Total protein extracts were analyzed for p53 and Ser15-phosphorylated p53 by western blotting. b COV434 Mock and COV434-FGF1 cells were treated or not with etoposide for 1, 2, 3 or 16 h. Total proteins were analyzed for PUMA, Bax and p21 protein levels by western blotting. c Average apoptosis rates ± SEM for 2 experiments done in triplicate measured by flow cytometry of non-transfected COV434 (NT), Mock and FGF1 cells treated with etoposide for 16 h. Cells were pretreated or not for 90 min with the p53 transcriptional inhibitor pifithrin-alpha (PFT-α, 30 µM). d Western blot analysis of p21 and Bax protein levels in COV434 Mock and FGF1 cells pretreated with PFT-α (30 µM for 90 min), followed by an etoposide (25 µg/mL) treatment for 6 or 16 h. Two-tailed t -tests results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: p53 transcriptional-dependent activities are dispensable for the apoptosis of the COV434 ovarian cell line. a COV434-Mock and COV434-FGF1 cells were treated or not with 25 µg/mL etoposide for 1, 2, 3 or 16 h. Total protein extracts were analyzed for p53 and Ser15-phosphorylated p53 by western blotting. b COV434 Mock and COV434-FGF1 cells were treated or not with etoposide for 1, 2, 3 or 16 h. Total proteins were analyzed for PUMA, Bax and p21 protein levels by western blotting. c Average apoptosis rates ± SEM for 2 experiments done in triplicate measured by flow cytometry of non-transfected COV434 (NT), Mock and FGF1 cells treated with etoposide for 16 h. Cells were pretreated or not for 90 min with the p53 transcriptional inhibitor pifithrin-alpha (PFT-α, 30 µM). d Western blot analysis of p21 and Bax protein levels in COV434 Mock and FGF1 cells pretreated with PFT-α (30 µM for 90 min), followed by an etoposide (25 µg/mL) treatment for 6 or 16 h. Two-tailed t -tests results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Transfection, Two Tailed Test

Both FGFR-dependent and FGFR-independent pathways are involved in FGF1 anti-apoptotic activity. a Western blot analysis for FGF1 levels in total extracts and conditioned media of non-transfected COV434 (NT), COV434-Mock and COV434-FGF1 cells. Endogenous FGF1 is detected in all total extracts whereas exogenous FGF1-V5-His is seen only in FGF1-overexpressing cells as expected. b Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock cells. Cells were pretreated or not with the FGFR1/3 inhibitor PD173074 (25 nM for one hour), followed or not by a treatment with 50 ng/mL of recombinant FGF1 (rFGF1) supplemented with 10 µg/mL heparin for 24 h. On the next day, these treatments were renewed adding or not etoposide for 16 h. c Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock and COV434-FGF1 cells pretreated or not with 25 nM PD173074 for 24 h, and treated or not with etoposide (25 µg/mL for 16 h). Two-tailed t -tests are indicated by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001
Figure Legend Snippet: Both FGFR-dependent and FGFR-independent pathways are involved in FGF1 anti-apoptotic activity. a Western blot analysis for FGF1 levels in total extracts and conditioned media of non-transfected COV434 (NT), COV434-Mock and COV434-FGF1 cells. Endogenous FGF1 is detected in all total extracts whereas exogenous FGF1-V5-His is seen only in FGF1-overexpressing cells as expected. b Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock cells. Cells were pretreated or not with the FGFR1/3 inhibitor PD173074 (25 nM for one hour), followed or not by a treatment with 50 ng/mL of recombinant FGF1 (rFGF1) supplemented with 10 µg/mL heparin for 24 h. On the next day, these treatments were renewed adding or not etoposide for 16 h. c Average apoptosis rates ± SEM for 3 experiments done in triplicate measured by flow cytometry of COV434-Mock and COV434-FGF1 cells pretreated or not with 25 nM PD173074 for 24 h, and treated or not with etoposide (25 µg/mL for 16 h). Two-tailed t -tests are indicated by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Techniques Used: Activity Assay, Western Blot, Transfection, Flow Cytometry, Cytometry, Recombinant, Two Tailed Test

15) Product Images from "Bid can mediate a pro-apoptotic response to etoposide and ionizing radiation without cleavage in its unstructured loop and in the absence of p53"

Article Title: Bid can mediate a pro-apoptotic response to etoposide and ionizing radiation without cleavage in its unstructured loop and in the absence of p53

Journal: Oncogene

doi: 10.1038/onc.2011.75

Bid-dependent apoptosis in response to etoposide and IR proceeds via the mitochondria. p53-RNAi Bid −/− MEFs that had been transduced to stably express WT Bid were used as such (control) or additionally transduced (Td) to express Bcl-2 or dnCasp-9. ( a , c ) Downregulation of p53 by RNAi and expression of Bid, Bcl-2 or dnCasp-9 protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) Control or Bcl-2-expressing cells were exposed to the indicated dosages of etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide) or 48 h (IR). ( d ) Control or dnCasp-9-expressing cells were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as indicated for panel ( b ). Data presented in ( b ) and ( d ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of control and Bcl-2 or dnCasp-9 samples are indicated for * P
Figure Legend Snippet: Bid-dependent apoptosis in response to etoposide and IR proceeds via the mitochondria. p53-RNAi Bid −/− MEFs that had been transduced to stably express WT Bid were used as such (control) or additionally transduced (Td) to express Bcl-2 or dnCasp-9. ( a , c ) Downregulation of p53 by RNAi and expression of Bid, Bcl-2 or dnCasp-9 protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) Control or Bcl-2-expressing cells were exposed to the indicated dosages of etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide) or 48 h (IR). ( d ) Control or dnCasp-9-expressing cells were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as indicated for panel ( b ). Data presented in ( b ) and ( d ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of control and Bcl-2 or dnCasp-9 samples are indicated for * P

Techniques Used: Stable Transfection, Expressing

Bid cleavage at aspartate residues in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, or the Bid mutants Bid D60E, Bid D75E, Bid D60E/D75E or Bid D55E/D60E/D75E. ( a , d ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid, Bid D60E or Bid D75E were exposed to the indicated dosages of TNFα+cycloheximide (CHX), etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 5 h (TNFα+CHX), 24 h (etoposide) or 48 h (IR). ( c ) P53-RNAi Bid −/− MEFs expressing WT Bid or Bid D60E/D75E were exposed to the indicated dosages of TNFα+CHX, etoposide or IR and apoptosis was read out as outlined for panel (b). ( e ) P53-RNAi Bid −/− MEFs expressing WT Bid or Bid D55E/D60E/D75E were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as outlined for panel ( b ). Data presented in ( b ), ( c ) and ( e ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of Bid and Bid D60E or D60E/D75E samples are indicated for * P
Figure Legend Snippet: Bid cleavage at aspartate residues in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, or the Bid mutants Bid D60E, Bid D75E, Bid D60E/D75E or Bid D55E/D60E/D75E. ( a , d ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid, Bid D60E or Bid D75E were exposed to the indicated dosages of TNFα+cycloheximide (CHX), etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 5 h (TNFα+CHX), 24 h (etoposide) or 48 h (IR). ( c ) P53-RNAi Bid −/− MEFs expressing WT Bid or Bid D60E/D75E were exposed to the indicated dosages of TNFα+CHX, etoposide or IR and apoptosis was read out as outlined for panel (b). ( e ) P53-RNAi Bid −/− MEFs expressing WT Bid or Bid D55E/D60E/D75E were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as outlined for panel ( b ). Data presented in ( b ), ( c ) and ( e ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of Bid and Bid D60E or D60E/D75E samples are indicated for * P

Techniques Used: Stable Transfection, Expressing, Mutagenesis

Bid can contribute to clonogenic cell death in response to etoposide and IR. ( a , b ) P53-RNAi Bid −/− MEFs carrying an empty vector (EV) or stably expressing WT Bid were exposed to the indicated dosages of etoposide ( a ) or IR ( b ). Surviving colonies were counted 14 days post-treatment. The relative surviving fraction is plotted against the dose of etoposide or IR. Data presented are representative of two independent experiments.
Figure Legend Snippet: Bid can contribute to clonogenic cell death in response to etoposide and IR. ( a , b ) P53-RNAi Bid −/− MEFs carrying an empty vector (EV) or stably expressing WT Bid were exposed to the indicated dosages of etoposide ( a ) or IR ( b ). Surviving colonies were counted 14 days post-treatment. The relative surviving fraction is plotted against the dose of etoposide or IR. Data presented are representative of two independent experiments.

Techniques Used: Plasmid Preparation, Stable Transfection, Expressing

Bid cleavage in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid or Bid with the unstructured loop exchanged for a random stretch of Gly and Ser amino acids (Bid w/o loop). ( a ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid or the Bid w/o loop mutant were exposed to the indicated dosages of TNFα+cycloheximide (CHX), etoposide or IR. After 5 h (TNFα+CHX), 24 h (etoposide) or 48 h (IR), apoptosis levels were determined as the percentage of cells with cleaved caspase-3. Data are expressed as means of three independent experiments±s.d. Statistically significant differences between values of Bid and Bid w/o loop are indicated for ** P
Figure Legend Snippet: Bid cleavage in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid or Bid with the unstructured loop exchanged for a random stretch of Gly and Ser amino acids (Bid w/o loop). ( a ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid or the Bid w/o loop mutant were exposed to the indicated dosages of TNFα+cycloheximide (CHX), etoposide or IR. After 5 h (TNFα+CHX), 24 h (etoposide) or 48 h (IR), apoptosis levels were determined as the percentage of cells with cleaved caspase-3. Data are expressed as means of three independent experiments±s.d. Statistically significant differences between values of Bid and Bid w/o loop are indicated for ** P

Techniques Used: Stable Transfection, Expressing, Mutagenesis

Bid uses its BH3 domain to indirectly activate Bax and/or Bak during etoposide- and IR-induced apoptosis. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, the Bid G94E mutant or the Bid G94A mutant. ( a , c ) Downregulation of p53 by RNAi and Bid protein expression from the introduced vectors were validated by immunoblotting, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid or Bid G94E were exposed to the indicated dosages of etoposide or IR, or to TNFα in combination with cycloheximide (CHX) as a validation of the Bid G94E mutant. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide), 48 h (IR) or 5 h (TNFα+CHX). Background apoptosis levels, as those observed in p53-RNAi Bid −/− MEF carrying EV2, were subtracted from obtained values for all stimuli. ( d ) p53-RNAi Bid −/− MEFs expressing WT Bid or Bid G94A were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as outlined in ( b ). Data in ( b ) and ( d ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of WT Bid and Bid G94E are indicated in ( b ) for * P
Figure Legend Snippet: Bid uses its BH3 domain to indirectly activate Bax and/or Bak during etoposide- and IR-induced apoptosis. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, the Bid G94E mutant or the Bid G94A mutant. ( a , c ) Downregulation of p53 by RNAi and Bid protein expression from the introduced vectors were validated by immunoblotting, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid or Bid G94E were exposed to the indicated dosages of etoposide or IR, or to TNFα in combination with cycloheximide (CHX) as a validation of the Bid G94E mutant. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide), 48 h (IR) or 5 h (TNFα+CHX). Background apoptosis levels, as those observed in p53-RNAi Bid −/− MEF carrying EV2, were subtracted from obtained values for all stimuli. ( d ) p53-RNAi Bid −/− MEFs expressing WT Bid or Bid G94A were exposed to the indicated dosages of etoposide or IR and apoptosis was read out as outlined in ( b ). Data in ( b ) and ( d ) are expressed as means of three independent experiments±s.d. Statistically significant differences between values of WT Bid and Bid G94E are indicated in ( b ) for * P

Techniques Used: Stable Transfection, Mutagenesis, Expressing

The p53 status can influence the reliance on Bid for etoposide- and IR-induced apoptosis. SV40-transformed Bid −/− MEFs were retrovirally transduced (Td) with an empty RNAi vector (EV1) or with p53 shRNA, together with an empty expression vector (EV2) or a vector encoding WT Bid. ( a ) Validation of p53 downregulation by p53 shRNA and Bid expression from the introduced vector in the relevant MEF cell lines, as demonstrated by immunoblotting with antibodies to p53 and Bid on total cell lysates. Immunoblotting for actin served as a loading control ( b , c ) Sensitivity of control (EV1-transduced) MEFs ( b ) and p53-deficient (p53-RNAi) MEFs ( c ) to apoptosis induction by DNA-damaging regimens. Cells were exposed to the indicated dosages of etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide) or 48 h (IR). Data presented are expressed as means of three independent experiments±s.d. Statistically significant differences between values of EV and Bid samples are indicated for * P
Figure Legend Snippet: The p53 status can influence the reliance on Bid for etoposide- and IR-induced apoptosis. SV40-transformed Bid −/− MEFs were retrovirally transduced (Td) with an empty RNAi vector (EV1) or with p53 shRNA, together with an empty expression vector (EV2) or a vector encoding WT Bid. ( a ) Validation of p53 downregulation by p53 shRNA and Bid expression from the introduced vector in the relevant MEF cell lines, as demonstrated by immunoblotting with antibodies to p53 and Bid on total cell lysates. Immunoblotting for actin served as a loading control ( b , c ) Sensitivity of control (EV1-transduced) MEFs ( b ) and p53-deficient (p53-RNAi) MEFs ( c ) to apoptosis induction by DNA-damaging regimens. Cells were exposed to the indicated dosages of etoposide or IR. Apoptosis levels were determined as the percentage of cells with cleaved caspase-3 after 24 h (etoposide) or 48 h (IR). Data presented are expressed as means of three independent experiments±s.d. Statistically significant differences between values of EV and Bid samples are indicated for * P

Techniques Used: Transformation Assay, Plasmid Preparation, shRNA, Expressing

Bid cleavage at alternative defined proteolytic cleavage sites in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, or the Bid Q58A, Bid S65A or Bid G70A/R71A mutants. ( a ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid, Bid Q58A, Bid S65A or Bid G70A/R71A were exposed to the indicated dosages of etoposide or IR. After 24 h (etoposide) or 48 h (IR), apoptosis levels were determined as the percentage of cells with cleaved caspase-3. Data are expressed as means of three independent experiments±s.d.
Figure Legend Snippet: Bid cleavage at alternative defined proteolytic cleavage sites in its unstructured loop is not required for apoptosis induction in response to etoposide and IR. p53-RNAi Bid −/− MEFs were transduced (Td) to stably express WT Bid, or the Bid Q58A, Bid S65A or Bid G70A/R71A mutants. ( a ) Downregulation of p53 by RNAi and expression of WT or mutant Bid protein from the introduced vectors were validated by immunoblotting on total cell lysates, where actin served as a loading control. ( b ) p53-RNAi Bid −/− MEFs expressing WT Bid, Bid Q58A, Bid S65A or Bid G70A/R71A were exposed to the indicated dosages of etoposide or IR. After 24 h (etoposide) or 48 h (IR), apoptosis levels were determined as the percentage of cells with cleaved caspase-3. Data are expressed as means of three independent experiments±s.d.

Techniques Used: Stable Transfection, Expressing, Mutagenesis

16) Product Images from "Glutathione-mediated antioxidant response and aerobic metabolism: two crucial factors involved in determining the multi-drug resistance of high-risk neuroblastoma"

Article Title: Glutathione-mediated antioxidant response and aerobic metabolism: two crucial factors involved in determining the multi-drug resistance of high-risk neuroblastoma

Journal: Oncotarget

doi: 10.18632/oncotarget.12209

HTLA-Chr cells are characterized by higher GSH levels, a lower amount of a lipid peroxidation marker and up-regulation of GST activity A. Reduced and oxidized glutathione (GSH and GSSG) levels were analyzed in untreated and in 1.25 μM etoposide-treated (24 hrs) HTLA and HTLA-Chr cells. Results were reported as μM/μg protein. Histogram summarizes quantitative data of means ± S.E.M. of six independent experiments. ** p
Figure Legend Snippet: HTLA-Chr cells are characterized by higher GSH levels, a lower amount of a lipid peroxidation marker and up-regulation of GST activity A. Reduced and oxidized glutathione (GSH and GSSG) levels were analyzed in untreated and in 1.25 μM etoposide-treated (24 hrs) HTLA and HTLA-Chr cells. Results were reported as μM/μg protein. Histogram summarizes quantitative data of means ± S.E.M. of six independent experiments. ** p

Techniques Used: Marker, Activity Assay

NAC treatment enhances GSH levels, decreases H 2 O 2 production and markedly promotes the tumorigenic potential of neuroblastoma cells A. GSH levels were analyzed in HTLA and HTLA-Chr cells treated with 2 mM NAC or pre-treated (1 hr) with 2 mM NAC and then exposed (24 hrs) to 1.25 μM etoposide. Histogram summarizes quantitative data of the means ± S.E.M. of three independent experiments. ** p
Figure Legend Snippet: NAC treatment enhances GSH levels, decreases H 2 O 2 production and markedly promotes the tumorigenic potential of neuroblastoma cells A. GSH levels were analyzed in HTLA and HTLA-Chr cells treated with 2 mM NAC or pre-treated (1 hr) with 2 mM NAC and then exposed (24 hrs) to 1.25 μM etoposide. Histogram summarizes quantitative data of the means ± S.E.M. of three independent experiments. ** p

Techniques Used:

HTLA-Chr cells develop a multi-drug resistant phenotype Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (1.25–100 μM) for 24 hrs A. of doxorubicin (0.046-14.72 μM) for 24, 48 and 72 hrs B. and of H 2 O 2 (250-1000 μM) for 3 hrs C. Histograms summarize quantitative data of the means ± S.E.M. of four independent experiments. * p
Figure Legend Snippet: HTLA-Chr cells develop a multi-drug resistant phenotype Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (1.25–100 μM) for 24 hrs A. of doxorubicin (0.046-14.72 μM) for 24, 48 and 72 hrs B. and of H 2 O 2 (250-1000 μM) for 3 hrs C. Histograms summarize quantitative data of the means ± S.E.M. of four independent experiments. * p

Techniques Used: MTT Assay

HTLA-Chr cells do not change H 2 O 2 production after treatment with etoposide or doxorubicin H 2 O 2 production was analyzed in HTLA and in HTLA-Chr cells incubated for 24 hrs with increasing concentrations of etoposide (1.25–100 μM) A. or doxorubicin (0.046-14.72 μM) B. Histograms summarize quantitative data of the means ± S.E.M. of three independent experiments. * p
Figure Legend Snippet: HTLA-Chr cells do not change H 2 O 2 production after treatment with etoposide or doxorubicin H 2 O 2 production was analyzed in HTLA and in HTLA-Chr cells incubated for 24 hrs with increasing concentrations of etoposide (1.25–100 μM) A. or doxorubicin (0.046-14.72 μM) B. Histograms summarize quantitative data of the means ± S.E.M. of three independent experiments. * p

Techniques Used: Incubation

HTLA-Chr cells have a major oxygen consumption, an increased oxidative phosphorylation and an up-regulation of catalase activity A. The oxygen consumption rate (OCR) was evaluated in untreated and in 1.25 μM etoposide-treated (24 hrs) HTLA and HTLA-Chr cells. Results were reported as nmol O 2 /min/10 6 cells. Histogram summarizes quantitative data of means ± S.E.M. of three independent experiments. ** p
Figure Legend Snippet: HTLA-Chr cells have a major oxygen consumption, an increased oxidative phosphorylation and an up-regulation of catalase activity A. The oxygen consumption rate (OCR) was evaluated in untreated and in 1.25 μM etoposide-treated (24 hrs) HTLA and HTLA-Chr cells. Results were reported as nmol O 2 /min/10 6 cells. Histogram summarizes quantitative data of means ± S.E.M. of three independent experiments. ** p

Techniques Used: Activity Assay

BSO treatment induces GSH depletion, increases H 2 O 2 production and markedly reduces the tumorigenic potential of etoposide-resistant cells A. GSH levels were analyzed in HTLA and HTLA-Chr cell treated with 1 mM BSO or pre-treated (1 hr) with 1 mM BSO and then exposed (24 hrs) to 1.25 μM etoposide. Results were reported as μM/μg protein. Histogram summarizes quantitative data of the means ± S.E.M. of three independent experiments. ** p
Figure Legend Snippet: BSO treatment induces GSH depletion, increases H 2 O 2 production and markedly reduces the tumorigenic potential of etoposide-resistant cells A. GSH levels were analyzed in HTLA and HTLA-Chr cell treated with 1 mM BSO or pre-treated (1 hr) with 1 mM BSO and then exposed (24 hrs) to 1.25 μM etoposide. Results were reported as μM/μg protein. Histogram summarizes quantitative data of the means ± S.E.M. of three independent experiments. ** p

Techniques Used:

Chronically-etoposide-treated HTLA cells (HTLA-Chr) are less proliferating and tumorigenic than untreated HTLA parental cells and they evade apoptotic death induced by etoposide exposure A. Proliferation assay. HTLA parental cells and HTLA-Chr cells were incubated with CFDA-SE and the intensity of cellular CFDA-SE fluorescence was evaluated at 24 hrs and 48 hrs after 1.25 μM etoposide treatment. Results were expressed as proliferation index and are the means ±S.E.M. of three independent experiments. ** p
Figure Legend Snippet: Chronically-etoposide-treated HTLA cells (HTLA-Chr) are less proliferating and tumorigenic than untreated HTLA parental cells and they evade apoptotic death induced by etoposide exposure A. Proliferation assay. HTLA parental cells and HTLA-Chr cells were incubated with CFDA-SE and the intensity of cellular CFDA-SE fluorescence was evaluated at 24 hrs and 48 hrs after 1.25 μM etoposide treatment. Results were expressed as proliferation index and are the means ±S.E.M. of three independent experiments. ** p

Techniques Used: Proliferation Assay, Incubation, Fluorescence

17) Product Images from "Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation"

Article Title: Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation

Journal: Scientific Reports

doi: 10.1038/s41598-018-32195-7

1p36 allelic loss is not observed in parental and HTLA-ER cells and miRNA-34a levels are reduced in both cell populations following etoposide treatment. ( A ) FISH analysis of HTLA-230 cells and HTLA-ER cells. Upper and lower left panels: metaphase of both HTLA-230 and HTLA-ER cells displays one normal chromosome 1 (close arrow) one 1 p arm derivative (close arrowhead) and 1 q arm derivative (open arrow); lower right panel: metaphase of HTLA-ER cells displaying one additional 1 p arm derivative (open arrowhead). ( B ) Expression levels of miRNA-34a in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: 1p36 allelic loss is not observed in parental and HTLA-ER cells and miRNA-34a levels are reduced in both cell populations following etoposide treatment. ( A ) FISH analysis of HTLA-230 cells and HTLA-ER cells. Upper and lower left panels: metaphase of both HTLA-230 and HTLA-ER cells displays one normal chromosome 1 (close arrow) one 1 p arm derivative (close arrowhead) and 1 q arm derivative (open arrow); lower right panel: metaphase of HTLA-ER cells displaying one additional 1 p arm derivative (open arrowhead). ( B ) Expression levels of miRNA-34a in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p

Techniques Used: Fluorescence In Situ Hybridization, Expressing

Parental and HTLA-ER cells express a non-inducible P53 protein carrying the homozygous TP53 missense mutation A161T. ( A ) Protein levels of P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. ( B ) Transactivation ability of wild-type and mutant (A161T) P53 proteins in yLFM-P21-5′, yLFM-BAX A + B and yLFM-MDM2P2C yeast strains. The transactivation ability was determined at two different temperatures (30 °C and 36 °C) using a constitutive expression of P53 proteins (ADH1 promoter). Presented data are the fold of induction over empty vector (pRS315) and standard deviation of four biological replicates. # p
Figure Legend Snippet: Parental and HTLA-ER cells express a non-inducible P53 protein carrying the homozygous TP53 missense mutation A161T. ( A ) Protein levels of P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. ( B ) Transactivation ability of wild-type and mutant (A161T) P53 proteins in yLFM-P21-5′, yLFM-BAX A + B and yLFM-MDM2P2C yeast strains. The transactivation ability was determined at two different temperatures (30 °C and 36 °C) using a constitutive expression of P53 proteins (ADH1 promoter). Presented data are the fold of induction over empty vector (pRS315) and standard deviation of four biological replicates. # p

Techniques Used: Mutagenesis, Western Blot, Expressing, Plasmid Preparation, Standard Deviation

P53 Ser15 phosphorylation is detected only in etoposide-treated HTLA parental cells and is associated with PPM1D up-regulation. ( A ) Protein levels of phospho-(Ser15)-P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of phospho-P53/P53 ratio means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: P53 Ser15 phosphorylation is detected only in etoposide-treated HTLA parental cells and is associated with PPM1D up-regulation. ( A ) Protein levels of phospho-(Ser15)-P53 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of phospho-P53/P53 ratio means ± S.E.M of three independent experiments. ** p

Techniques Used: Western Blot

HTLA-ER cells have a deletion at the 13q14.3 locus which is associated with decreased levels of miRNAs 15a/16-1 in respect to parental cells. ( A ) FISH analysis of HTLA-230 and HTLA-ER cells: Upper panels: nuclei of HTLA-230 cells with two CEP12, two 13q34 and two D13S319 signals; nuclei of HTLA-ER cells with two CEP12, three 13q34 and one D13S319 signals. Lower panels: metaphase of HTLA-230 cells with two chromosomes 13 displaying 13q34 and D13S319 signals; metaphase of HTLA-ER cells with one chromosome 13 displaying 13q34 and D13S319 signals, one rearranged chromosome 13 displaying two 13q34, and one chromosome 12 displaying cep 12signal. ( B ) Expression levels of miRNA-15a (left panel) and miRNA-16 (right panel) in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p
Figure Legend Snippet: HTLA-ER cells have a deletion at the 13q14.3 locus which is associated with decreased levels of miRNAs 15a/16-1 in respect to parental cells. ( A ) FISH analysis of HTLA-230 and HTLA-ER cells: Upper panels: nuclei of HTLA-230 cells with two CEP12, two 13q34 and two D13S319 signals; nuclei of HTLA-ER cells with two CEP12, three 13q34 and one D13S319 signals. Lower panels: metaphase of HTLA-230 cells with two chromosomes 13 displaying 13q34 and D13S319 signals; metaphase of HTLA-ER cells with one chromosome 13 displaying 13q34 and D13S319 signals, one rearranged chromosome 13 displaying two 13q34, and one chromosome 12 displaying cep 12signal. ( B ) Expression levels of miRNA-15a (left panel) and miRNA-16 (right panel) in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.E.M of three independent experiments. ** p

Techniques Used: Fluorescence In Situ Hybridization, Expressing

The mitotic index of HTLA-ER cells and their Bax/Bcl2 ratio were not modified by acute etoposide exposure. ( A ) Mitotic index of HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.D. of four independent experiments per experimental condition (at least 4 × 10 3 cells per experimental condition were counted) ** p
Figure Legend Snippet: The mitotic index of HTLA-ER cells and their Bax/Bcl2 ratio were not modified by acute etoposide exposure. ( A ) Mitotic index of HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Histograms summarize quantitative data of means ± S.D. of four independent experiments per experimental condition (at least 4 × 10 3 cells per experimental condition were counted) ** p

Techniques Used: Modification

BMI-1 overexpression with consequent p16 down-regulation is found in HTLA-ER cells in respect to parental cells. ( A ) Protein levels of BMI-1 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of protein expression levels means, normalized to β-actin expression ± S.E.M of three independent experiments. * p
Figure Legend Snippet: BMI-1 overexpression with consequent p16 down-regulation is found in HTLA-ER cells in respect to parental cells. ( A ) Protein levels of BMI-1 in HTLA-230 and HTLA-ER cells untreated or treated for 24 hrs with 1.25 μM etoposide. Immunoblots are representative of three independent experiments with essentially similar results. β-Actin is the internal loading control. Histograms summarize quantitative data of protein expression levels means, normalized to β-actin expression ± S.E.M of three independent experiments. * p

Techniques Used: Over Expression, Western Blot, Expressing

18) Product Images from "Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage"

Article Title: Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

Journal: Molecular Cell

doi: 10.1016/j.molcel.2018.07.034

cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. Data are presented as mean values of biological triplicates ± SD. See also Figure S4 .
Figure Legend Snippet: cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. Data are presented as mean values of biological triplicates ± SD. See also Figure S4 .

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Translocation Assay, Liquid Chromatography with Mass Spectroscopy

Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. Data are presented as mean values of biological triplicates ± SD. See also Figure S5 .
Figure Legend Snippet: Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. Data are presented as mean values of biological triplicates ± SD. See also Figure S5 .

Techniques Used: Activation Assay, Activity Assay, Transfection, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. Data are presented as mean values of biological triplicates ± SD. See also Figures S2 and S3 A–S3F.
Figure Legend Snippet: STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. Data are presented as mean values of biological triplicates ± SD. See also Figures S2 and S3 A–S3F.

Techniques Used: Expressing, Clonogenic Cell Survival Assay, Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Translocation Assay, Reverse Transcription Polymerase Chain Reaction

TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. See also Figure S7 .
Figure Legend Snippet: TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. See also Figure S7 .

Techniques Used: Immunoprecipitation, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Molecular Weight, Modification, SDS Page

The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. Data are presented as mean values of biological triplicates ± SD. See also Figures S3 G–S3L.
Figure Legend Snippet: The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. Data are presented as mean values of biological triplicates ± SD. See also Figures S3 G–S3L.

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Translocation Assay, Luciferase

Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). Data are presented as mean values of biological triplicates ± SD. See also Figure S1 .
Figure Legend Snippet: Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). Data are presented as mean values of biological triplicates ± SD. See also Figure S1 .

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Cytotoxicity Assay, Expressing

Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). See also Figure S6 .
Figure Legend Snippet: Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). See also Figure S6 .

Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Quantitative RT-PCR

19) Product Images from "Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage"

Article Title: Non-canonical Activation of the DNA Sensing Adaptor STING by ATM and IFI16 Mediates NF-κB Signaling after Nuclear DNA Damage

Journal: Molecular Cell

doi: 10.1016/j.molcel.2018.07.034

cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .
Figure Legend Snippet: cGAS Is Dispensable for the Early Innate Immune Response to Nuclear DNA Damage (A) Immunoblotting analysis of WT and two cGAS −/− HaCaT clones treated with DMSO or 50 μM etoposide for 6 hr. (B and C) WT and cGAS −/− HaCaT cells were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (B) and IL-6 (C) mRNA expression. (D) IL-6 in supernatants from WT and cGAS −/− HaCaT cells treated with 50 μM etoposide quantified by ELISA. (E) MRC-5 fibroblasts were treated with non-targeting (NT) or cGAS -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr. cGAS protein expression was analyzed by western blot. (F) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 fibroblasts treated with siRNA as in (E) and stimulated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA for 6 hr. (G) PMA-differentiated WT, cGAS −/− , and IFI16 −/− THP1 cells were treated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (H) WT and cGAS −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr, stained for p65 (green) and DNA (DAPI, blue), and visualized by confocal microscopy. Scale bar, 20 μm. (I) Quantification of p65 translocation from (H). (J) HaCaT cells were treated with 50 μM etoposide for the indicated times or transfected with 1 μg/mL HT-DNA for 4 hr. cGAMP production was quantified by LC-MS. .

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Translocation Assay, Liquid Chromatography with Mass Spectroscopy

Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .
Figure Legend Snippet: Etoposide-Induced NF-κB Activation Involves DNA Damage Factors, but Not TBK1 Activity (A) HaCaT cells grown on coverslips were pre-treated for 30 min with 3 μg/mL brefeldin A where indicated before stimulation with 50 μM etoposide or transfection of 1 μg/mL HT-DNA. Cells were fixed and stained for STING (green) and DNA (DAPI, blue). Scale bar, 20 μm. (B and C) HaCaT cells were pre-treated for 30 min with 3 μg/mL brefeldin A before treatment with 50 μM etoposide or DMSO, mock transfection (Lipo), or transfection of 1 μg/mL HT-DNA for 6 hr. IFN-β (B) and IL-6 (C) mRNA expression was analyzed by qRT-PCR. (D and E) HaCaT cells were pre-treated for 1 hr with 2 μM TBK1 inhibitor MRT67307 and stimulated as in (B) before qRT-PCR analysis of IFN-β (D) and IL-6 (E) mRNA expression. (F) HaCaT cells grown on coverslips were pre-treated with 2 μM TBK1 inhibitor MRT67307 for 1 hr before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (G and H) HaCaT cells were pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 and stimulated as in (B). IFN-β (G) and IL-6 (H) mRNA expression was quantified by qRT-PCR. (I) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (G) and stimulated for 24 hr. (J) HaCaT cells grown on coverslips were pre-treated for 1 hr with 10 μM KU55933 before 4 hr of stimulation with 50 μM etoposide. Cells were fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (K) qRT-PCR analysis of IFN-β mRNA expression in NHEK cells pre-treated for 1 hr with 10 μM KU55933, followed by treatment with 50 μM etoposide for 24 hr. (L) qRT-PCR analysis of IFN-β mRNA in HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34 before treatment as in (B) for 6 hr. .

Techniques Used: Activation Assay, Activity Assay, Transfection, Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.
Figure Legend Snippet: STING Is Required for the Innate Immune Response to Etoposide-Induced DNA Damage (A) Wild-type (WT) and STING −/− HaCaT cells were treated with DMSO or 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (B) Clonogenic survival assay of WT and STING −/− HaCaT cells. Numbers of colonies > 50 cells were counted and expressed as a percentage of untreated control. (C and D) WT HaCaT and two STING −/− clones were treated with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C) for 6 hr before qRT-PCR analysis of IFN-β (C) and IL-6 (D) mRNA expression. (E) ELISA analysis of IL-6 secretion in supernatants from cells treated as in (C) for 24 hr. (F) qRT-PCR array analysis of cytokine and chemokine expression in WT and STING −/− HaCaT cells treated with DMSO, 50 μM etoposide, Lipofectamine, or 1 μg/mL HT-DNA for 6 hr. Shown are genes induced at least 2-fold over controls. (G and H) WT and STING −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and stained for NF-κB p65 (green) and DNA (DAPI, blue) for analysis by confocal microscopy (G) and quantification of p65 nuclear translocation (H). Scale bar, 20 μm. (I and J) NHEKs were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 24 hr. STING protein levels were analyzed by immunoblotting (I), and IFN-β mRNA expression was quantified by qRT-PCR (J). (K) MRC-5 fibroblasts were treated with non-targeting (NT) or STING -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide for 6 hr and analysis of IFN-β mRNA by RT-PCR. (L) PMA-differentiated WT and STING −/− THP1 cells were stimulated with 50 μM etoposide for 30 hr or 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. A–S3F.

Techniques Used: Expressing, Clonogenic Cell Survival Assay, Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Translocation Assay, Reverse Transcription Polymerase Chain Reaction

TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .
Figure Legend Snippet: TRAF6 Mediates the K63-Linked Poly-ubiquitylation of STING (A) Immunoprecipitation of TRAF6 and STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. Immunoprecipitates (IP) with immunoglobulin G (IgG) control and input lysates were analyzed by immunoblotting. (B) WT and two TRAF6 −/− HaCaT clones were treated with 50 μM etoposide for 6 hr, and protein expression was analyzed by immunoblotting. (C) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (B). (D) WT and TRAF6 −/− HaCaT cells were treated with 50 μM etoposide or DMSO, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA for 6 hr before qRT-PCR analysis of IFN-β mRNA. (E) Immunoblotting analysis of WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the indicated times. (F) HaCaT cells were pre-treated for 1 hr with the indicated concentrations of Ubc13 inhibitor NSC697923 (NSC) before 6 hr of stimulation with 50 μM etoposide. IL-6 mRNA expression was quantified by qRT-PCR. (G) HEK293T cells were transfected with plasmids for the expression of IFI16, FLAG-tagged TRAF6, and hemagglutinin (HA)-tagged ubiquitin as indicated. 24 hr after transfection, STING was immunoprecipitated, and proteins in immunoprecipitates and input lysates were analyzed by immunoblotting. (H) Immunoprecipitation of K63-linked ubiquitin chains from WT and TRAF6 −/− HaCaT cells treated with 50 μM etoposide for the times indicated. Higher molecular weight forms of modified STING are visualized by gradient SDS-PAGE above the antibody heavy chain ( ∗ ), top panel, together with the association of unmodified STING, lower panel. .

Techniques Used: Immunoprecipitation, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Molecular Weight, Modification, SDS Page

The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.
Figure Legend Snippet: The Innate Immune Response to Etoposide-Induced Damage Involves IFI16 (A) Immunoblotting analysis of WT and IFI16 −/− HaCaT cells stimulated with 50 μM etoposide or DMSO for 6 hr. (B and C) WT HaCaT cells and two IFI16 −/− cell clones were treated for 6 hr with DMSO or 50 μM etoposide, mock transfected (Lipo), or transfected with 1 μg/mL HT-DNA or 100 ng/mL poly(I:C). IFN-β (B) or IL-6 (C) mRNA was quantified by qRT-PCR. (D) ELISA analysis of IL-6 protein in supernatants from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide for indicated times. (E) qRT-PCR analysis of CCL20 mRNA in WT and IFI16 −/− HaCaT cells treated with DMSO or 50 μM etoposide for 6 hr. (F) WT and IFI16 −/− HaCaT cells were treated as in (B) for 4 hr before analysis of protein expression by immunoblotting. (G) WT and IFI16 −/− HaCaT cells grown on coverslips were treated with 50 μM etoposide for 4 hr and fixed and stained for p65 (green) and DNA (DAPI, blue). Scale bar, 20 μm. (H) Quantification of p65 nuclear translocation in cells from (G). (I) Immunoblotting analysis of WT HaCaT cells and IFI16 −/− HaCaT cells reconstituted with lentiviruses for the expression of Luciferase (luc) or IFI16 as indicated. Cells were treated with doxycycline for 24 hr to induce expression and then stimulated with 50 μM etoposide for 6 hr. (J) qRT-PCR analysis of IFN-β mRNA in cells treated as in (I) as indicated. (K–M) MRC-5 fibroblasts treated with non-targeting (NT) or IFI16 -targeting siRNA pools for 48 hr before treatment with 50 μM etoposide or DMSO for 6 hr. IFI16 protein expression was analyzed by immunoblotting (K). IFN-β (L) and IL-6 (M) mRNA levels were analyzed by qRT-PCR. G–S3L.

Techniques Used: Clone Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Translocation Assay, Luciferase

Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .
Figure Legend Snippet: Etoposide-Mediated DNA Damage Induces an Acute Innate Immune Response in Human Cells (A–C) HaCaT keratinocytes were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (A), IL-6 (B), and CCL20 (C) mRNA. (D and E) Supernatants from cells treated with 50 μM etoposide were analyzed for secreted type I IFN using a bio-assay (D) or IL-6 protein using ELISA (E). (F) HaCaT cells were treated with 50 μM etoposide for the times indicated or transfected with 1 μg/mL herring testis (HT)-DNA for 6 hr. Phosphorylation of γH2A.X was analyzed by immunoblotting. (G) Cytotoxicity assay of HaCaT cells treated with 50 μM etoposide for the times indicated or lysed (Lys). (H and I) Primary normal human epidermal keratinocytes (NHEKs) from adult donors were treated with 50 μM etoposide for the times indicated before qRT-PCR analysis of IFN-β (H) and IL-6 (I) mRNA. (J) Supernatants from NHEK cells treated as in (H) were analyzed for IL-6 secretion by ELISA. (K) Cytotoxicity assay of NHEK cells treated as in (H) or lysed (Lys). (L) Primary MRC-5 fibroblasts were treated with 50 μM etoposide before qRT-PCR analysis of IFN-β mRNA expression. (M) Cytotoxicity assay of MRC-5 cells treated with 50 μM etoposide or lysed (Lys). (N) PMA-differentiated THP1 cells were stimulated with 50 μM etoposide for indicated times before qRT-PCR analysis of IFN-β mRNA. (O) Cytotoxicity assay of THP1 cells treated as in (N) or lysed (Lys). .

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Cytotoxicity Assay, Expressing

Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .
Figure Legend Snippet: Nuclear DNA Damage Results in the Assembly of a Non-canonical Signaling Complex Containing STING (A) Immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide for the indicated times. Immunoprecipitates (IPs) and whole-cell lysates were analyzed by immunoblotting. (B) Immunoblotting analysis following immunoprecipitation of STING from HaCaT cells treated with 50 μM etoposide or transfected with 1 μg/mL HT-DNA as indicated. (C) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM PARP inhibitor PJ34, followed by treatment with 50 μM etoposide for 2 hr. (D) Immunoprecipitation of STING from HaCaT cells pre-treated for 1 hr with 10 μM ATM inhibitor KU55933 followed by treatment with 50 μM etoposide. (E) Immunoprecipitation of STING from WT and IFI16 −/− HaCaT cells treated with 50 μM etoposide as indicated. (F) Immunoprecipitation of IFI16 from WT and STING −/− HaCaT cells treated with 50 μM etoposide as indicated. (G) HEK293T cells transfected with expression constructs for IFI16 and WT p53 or the S15A or S15D p53 mutants as indicated. 24 hr after transfection, IFI16 was immunoprecipitated from lysates. (H) p53 protein levels in HaCaT cells transfected with a non-targeting (NT) or a p53 -targeting siRNA pool for 48 hr before stimulation with 50 μM etoposide for 6 hr. (I) qRT-PCR analysis of IL-6 mRNA expression in cells treated as in (H). .

Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Quantitative RT-PCR

20) Product Images from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"

Article Title: Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines

Journal: Molecular Cancer

doi: 10.1186/1476-4598-6-61

Effect of hypoxia on the etoposide-induced p53 activation. A549, MCF-7 or HepG2 cells were incubated under normoxic or hypoxic conditions with or without etoposide (50 μM) for 1 or 16 hours. A , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-p53 antibody. Results are expressed in absorbance (means ± 1 SD, n = 3). B , Cells were cotransfected with the reporter plasmid pG13-Luc encoding the firefly luciferase and the pCMVβ normalization plasmid before being incubated 16 hours under normoxia (N) or hypoxia (H) in the presence or absence of etoposide (e, 50 μM). Results are expressed as means of the ratio between firefly luciferase activity and the β-galactosidase activity ± 1 SD (n = 3).
Figure Legend Snippet: Effect of hypoxia on the etoposide-induced p53 activation. A549, MCF-7 or HepG2 cells were incubated under normoxic or hypoxic conditions with or without etoposide (50 μM) for 1 or 16 hours. A , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-p53 antibody. Results are expressed in absorbance (means ± 1 SD, n = 3). B , Cells were cotransfected with the reporter plasmid pG13-Luc encoding the firefly luciferase and the pCMVβ normalization plasmid before being incubated 16 hours under normoxia (N) or hypoxia (H) in the presence or absence of etoposide (e, 50 μM). Results are expressed as means of the ratio between firefly luciferase activity and the β-galactosidase activity ± 1 SD (n = 3).

Techniques Used: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Activity Assay

Gene expression profiling, for genes involved in regulating cell cycle, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions. Please refer to supplementary data [Additional file 2 ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column.
Figure Legend Snippet: Gene expression profiling, for genes involved in regulating cell cycle, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions. Please refer to supplementary data [Additional file 2 ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column.

Techniques Used: Expressing, Incubation, RNA Extraction, Hybridization

Effect of hypoxia and/or etoposide on p21 (A), Bak, Bax and mdm2 (B) protein levels. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. p21, Bak, Bax and mdm2 were detected in total cell extracts by western blotting, using specific antibodies. a-tubulin was used to assess the total amount of proteins loaded on the gel.
Figure Legend Snippet: Effect of hypoxia and/or etoposide on p21 (A), Bak, Bax and mdm2 (B) protein levels. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. p21, Bak, Bax and mdm2 were detected in total cell extracts by western blotting, using specific antibodies. a-tubulin was used to assess the total amount of proteins loaded on the gel.

Techniques Used: Incubation, Western Blot

Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A , PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).
Figure Legend Snippet: Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A , PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).

Techniques Used: Incubation, Western Blot

Effect of hypoxia and/or etoposide on the HIF-1α protein level and HIF-1 DNA binding activity. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. A , HIF-1α was detected in total cell extracts by western blotting. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance (means ± 1 SD, n = 3).
Figure Legend Snippet: Effect of hypoxia and/or etoposide on the HIF-1α protein level and HIF-1 DNA binding activity. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. A , HIF-1α was detected in total cell extracts by western blotting. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance (means ± 1 SD, n = 3).

Techniques Used: Binding Assay, Activity Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Effect of hypoxia on the etoposide-induced p53 stabilization. A549, MCF-7 or HepG2 cells were incubated under normoxic or hypoxic conditions with or without etoposide (50 μM) for 1 or 16 hours. A , p53 was detected in total cell extracts by western blotting, using two specific anti-p53 antibodies (a monoclonal and a polyclonal). Phosphorylation of p53 on serine15 was revealed using a specific antibody. ERK2 and a-tubulin were used to assess the total amount of proteins loaded on the gel. ND = non detected B , after the incubation, cells were fixed, permeabilized and stained for p53 using a specific antibody (green). Nuclei were detected with To-Pro-3 (blue). Observation was performed using a confocal microscope with the photomultiplier constant.
Figure Legend Snippet: Effect of hypoxia on the etoposide-induced p53 stabilization. A549, MCF-7 or HepG2 cells were incubated under normoxic or hypoxic conditions with or without etoposide (50 μM) for 1 or 16 hours. A , p53 was detected in total cell extracts by western blotting, using two specific anti-p53 antibodies (a monoclonal and a polyclonal). Phosphorylation of p53 on serine15 was revealed using a specific antibody. ERK2 and a-tubulin were used to assess the total amount of proteins loaded on the gel. ND = non detected B , after the incubation, cells were fixed, permeabilized and stained for p53 using a specific antibody (green). Nuclei were detected with To-Pro-3 (blue). Observation was performed using a confocal microscope with the photomultiplier constant.

Techniques Used: Incubation, Western Blot, Staining, Microscopy

Gene expression profiling, for genes involved in regulating apoptosis, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions. Please refer to supplementary data [Additional file 2 ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column.
Figure Legend Snippet: Gene expression profiling, for genes involved in regulating apoptosis, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions. Please refer to supplementary data [Additional file 2 ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column.

Techniques Used: Expressing, Incubation, RNA Extraction, Hybridization

Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. A , procaspase 3 and the 20 kDa long subunit were detected in total cell extracts by western blotting, using a specific anti-caspase 3 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , the caspase 3 activity was assayed by measuring free AFC released from the cleavage of the caspase 3 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). C , PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel.
Figure Legend Snippet: Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. A , procaspase 3 and the 20 kDa long subunit were detected in total cell extracts by western blotting, using a specific anti-caspase 3 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B , the caspase 3 activity was assayed by measuring free AFC released from the cleavage of the caspase 3 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). C , PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel.

Techniques Used: Incubation, Western Blot, Activity Assay, Fluorescence

21) Product Images from "HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs)"

Article Title: HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs)

Journal: Oncotarget

doi: 10.18632/oncotarget.13807

Identification of the genes regulated in BJ-hTERT human fibroblasts by HIC1 in the presence and absence of etoposide to induce irreparable DSB ( A ) Schematic drawing of the experimental design of the study and of the 2 normalization strategies used to identify the genes regulated by HIC1 in the presence and absence of a 6 hours etoposide treatment to induce irreparable DSBs. Four experimental conditions were used and compared using two different comparison strategies. In the first comparison strategy (Strategy #1 : normalization of IV to III, shown as black lines), BJ-hTERT siCtrl cells treated with etoposide were compared to control cells (siCtrl, no etoposide) to define genes repressed by etoposide and thus containing a subset of genes repressed by SUMOylated HIC1. Then, BJ-hTERT siHIC1 cells treated or not with etoposide were compared to define genes still repressed by etoposide in a HIC1-deficient context. Subtracting [IV] from [II] yields 629 target genes repressed in response to DSBs and dependent upon HIC1 SUMOylation. In the second strategy (Strategy #2: normalization of IV to I, shown as dotted lines), BJ-hTERT siCtrl and BJ-hTERT siHIC1 cells, both treated with etoposide, were each compared to control cells (siCtrl, no etoposide). In that case, subtracting [IV] from [II] yields 475 target genes. ( B ) Venn diagrams showing the overlap of the 629 and 475 target genes from the 2 normalization strategies used yields a strong overlap of 319 genes (see text for detail).
Figure Legend Snippet: Identification of the genes regulated in BJ-hTERT human fibroblasts by HIC1 in the presence and absence of etoposide to induce irreparable DSB ( A ) Schematic drawing of the experimental design of the study and of the 2 normalization strategies used to identify the genes regulated by HIC1 in the presence and absence of a 6 hours etoposide treatment to induce irreparable DSBs. Four experimental conditions were used and compared using two different comparison strategies. In the first comparison strategy (Strategy #1 : normalization of IV to III, shown as black lines), BJ-hTERT siCtrl cells treated with etoposide were compared to control cells (siCtrl, no etoposide) to define genes repressed by etoposide and thus containing a subset of genes repressed by SUMOylated HIC1. Then, BJ-hTERT siHIC1 cells treated or not with etoposide were compared to define genes still repressed by etoposide in a HIC1-deficient context. Subtracting [IV] from [II] yields 629 target genes repressed in response to DSBs and dependent upon HIC1 SUMOylation. In the second strategy (Strategy #2: normalization of IV to I, shown as dotted lines), BJ-hTERT siCtrl and BJ-hTERT siHIC1 cells, both treated with etoposide, were each compared to control cells (siCtrl, no etoposide). In that case, subtracting [IV] from [II] yields 475 target genes. ( B ) Venn diagrams showing the overlap of the 629 and 475 target genes from the 2 normalization strategies used yields a strong overlap of 319 genes (see text for detail).

Techniques Used:

HIC1 and MTA1 recruitment to the HIC1-response elements in the SIRT1 promoter is increased upon induction of non-repairable DSBs ( A ) HIC1 recruitment to the HiRE in the SIRT1 promoter in various conditions. Chromatin was prepared from BJ-hTERT fibroblasts not-treated, mock-treated with DMSO or treated with 80 μM etoposide for various times and ChIP experiments were performed with antibodies against HIC1 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. ( B ) Etoposide-induced irreparable DSBs lead to an increase of HIC1 and MTA1 recruitment to the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-TERT fibroblasts mock-treated with DMSO or treated with 20 mM etoposide for 16 hours to induce irreparable DSBs and ChIP experiments were performed with antibodies against HIC1, MTA1 or rabbit IgG as described in panel A). Values that are statistically significantly different are indicated by bars and asterisks as follows: *P
Figure Legend Snippet: HIC1 and MTA1 recruitment to the HIC1-response elements in the SIRT1 promoter is increased upon induction of non-repairable DSBs ( A ) HIC1 recruitment to the HiRE in the SIRT1 promoter in various conditions. Chromatin was prepared from BJ-hTERT fibroblasts not-treated, mock-treated with DMSO or treated with 80 μM etoposide for various times and ChIP experiments were performed with antibodies against HIC1 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. ( B ) Etoposide-induced irreparable DSBs lead to an increase of HIC1 and MTA1 recruitment to the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-TERT fibroblasts mock-treated with DMSO or treated with 20 mM etoposide for 16 hours to induce irreparable DSBs and ChIP experiments were performed with antibodies against HIC1, MTA1 or rabbit IgG as described in panel A). Values that are statistically significantly different are indicated by bars and asterisks as follows: *P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

HIC1 SUMOylation is not required for efficient DSBs repair ( A ) Cos7 cells were transfected for 48 hours with wt FLAG-HIC1 or with the empty pcDNA3FLAG expression vector. Cells were either mock-treated with DMSO (–) or treated with 20 μM etoposide (+) for 1 hour. After removal of the drug, cells were allowed to recover in normal medium for various times (2, 4, 6 and 24 hours) and DSBs were monitored by neutral Comet assay. The percentage of Comet positive cells reflecting unrepaired DNA breaks is depicted after counting at least 100 cells in each condition. ( B ) Cos7 cells were transfected for 48 hours with wt FLAG-HIC1 or with the non-SUMOylatable E316A point mutant. Neutral Comet assays were performed and analyzed as described in panel A). The error bar indicates mean +/– standard deviation of three independent experiments (NS: not significant). ( C ) Representative Comet images of mock-treated (DMSO) and of cells treated with etoposide for 1 hour after transfection of wt HIC1 or of E316A HIC1 with or without recovery in normal medium for 4 and 24 hours, respectively. ( D ) Western blot analyses of cells transfected with wt HIC1 or with E316A HIC1 Samples of cells in each condition were taken before the Comet assays and immediately lysed in Laemmli loading buffer. These whole cell extracts were analyzed by Western blot with anti-FLAG antibodies to detect HIC1 and its SUMOylated forms. γH2AX and actin levels were used as controls for DSB induction and equal loading, respectively.
Figure Legend Snippet: HIC1 SUMOylation is not required for efficient DSBs repair ( A ) Cos7 cells were transfected for 48 hours with wt FLAG-HIC1 or with the empty pcDNA3FLAG expression vector. Cells were either mock-treated with DMSO (–) or treated with 20 μM etoposide (+) for 1 hour. After removal of the drug, cells were allowed to recover in normal medium for various times (2, 4, 6 and 24 hours) and DSBs were monitored by neutral Comet assay. The percentage of Comet positive cells reflecting unrepaired DNA breaks is depicted after counting at least 100 cells in each condition. ( B ) Cos7 cells were transfected for 48 hours with wt FLAG-HIC1 or with the non-SUMOylatable E316A point mutant. Neutral Comet assays were performed and analyzed as described in panel A). The error bar indicates mean +/– standard deviation of three independent experiments (NS: not significant). ( C ) Representative Comet images of mock-treated (DMSO) and of cells treated with etoposide for 1 hour after transfection of wt HIC1 or of E316A HIC1 with or without recovery in normal medium for 4 and 24 hours, respectively. ( D ) Western blot analyses of cells transfected with wt HIC1 or with E316A HIC1 Samples of cells in each condition were taken before the Comet assays and immediately lysed in Laemmli loading buffer. These whole cell extracts were analyzed by Western blot with anti-FLAG antibodies to detect HIC1 and its SUMOylated forms. γH2AX and actin levels were used as controls for DSB induction and equal loading, respectively.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Neutral Comet Assay, Mutagenesis, Standard Deviation, Western Blot

The SUMOylation increase of HIC1 upon induction of irreparable DSBs is dependent on ATM but independent of DNA-PKcs ( A ) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), either with a pool of four siRNAs targeting ATM (siATM) or with a pool of four siRNAs targeting DNAPKcs (siDNAPKcs). The next day, these cells were transfected with a FLAG-HIC1 expression vector for 24 hours and were then treated with 20 μM etoposide (+) or mock-treated with DMSO (–) as control for 16 hours before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) Quantification of SUMO-HIC1. The HIC1 SUMOylated band in control conditions (siCtrl, DMSO 16 h; lane 1 in panel A) was quantified with the Fujifilm MultiGauge software and given the arbitrary value of 1. The other HIC1 SUMOylated bands (lanes 2 to 6 in panel A) were quantified relative to this value. ( C ) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), a pool of four siRNAs targeting ATM (siATM) or with each individual siRNA from the pool targeting DNA-PKcs (siDNA-PKcs). Then, cells were treated with etoposide and total cell extracts were analyzed by Western blot on three different gels (two 6% polyacrylamide gels for DNAPK-cs and ATM; a 15% polyacrylamide gel for γH2AX, H2AX and actin) as described in panel A.
Figure Legend Snippet: The SUMOylation increase of HIC1 upon induction of irreparable DSBs is dependent on ATM but independent of DNA-PKcs ( A ) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), either with a pool of four siRNAs targeting ATM (siATM) or with a pool of four siRNAs targeting DNAPKcs (siDNAPKcs). The next day, these cells were transfected with a FLAG-HIC1 expression vector for 24 hours and were then treated with 20 μM etoposide (+) or mock-treated with DMSO (–) as control for 16 hours before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) Quantification of SUMO-HIC1. The HIC1 SUMOylated band in control conditions (siCtrl, DMSO 16 h; lane 1 in panel A) was quantified with the Fujifilm MultiGauge software and given the arbitrary value of 1. The other HIC1 SUMOylated bands (lanes 2 to 6 in panel A) were quantified relative to this value. ( C ) HEK293T cells were transfected either with nontargeted control siRNA (siCtrl), a pool of four siRNAs targeting ATM (siATM) or with each individual siRNA from the pool targeting DNA-PKcs (siDNA-PKcs). Then, cells were treated with etoposide and total cell extracts were analyzed by Western blot on three different gels (two 6% polyacrylamide gels for DNAPK-cs and ATM; a 15% polyacrylamide gel for γH2AX, H2AX and actin) as described in panel A.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Software

The increase of HIC1 SUMOylation upon irreparable DSB induction by etoposide requires ATM but not its effector kinase Chk2 ( A ) HEK293T cells were transfected with the FLAG and FLAG-HIC1 vectors. 48 hours after transfection, cells were pre-incubated or not with the Chk2 inhibitor (Chk2i) for 1 hour and then with etoposide for 16 hours as indicated. Cell extracts were prepared and Western blotting was performed with the indicated antibodies. *refers to non-specific bands detected by the anti pS516Chk2 (autophosphoylation) and by the anti pS20P53 (Chk2 target) antibodies. γH2AX and actin levels were used as controls for DSBs induction and equal loading, respectively. ( B ) To control for the inhibition of Chk2, HEK293T cells were transfected and pre-incubated or not with the Chk2 inhibitor (Chk2i) for 1 hour and then with etoposide for 16 hours exactly as in panel A) before lysis and Western blot analyses with the indicated antibodies ( C ) HEK293T cells grown in normal medium were transfected with siRNA control (siCtrl) or with a Chk2 siRNA pool (siChk2). Total RNAs were extracted and the mRNA expression levels of Chk2 were assessed by qRT-PCR. Values were normalized to 18S . ( D ) HEK293T cells were transfected either with non-target control siRNA (siCtrl) or with a Chk2 siRNA pool (siChk2) before being transfected with the indicated combination of FLAG, FLAG-HIC1 and SUMO-2 expression vectors. Cells were either incubated with DMSO (–) or with 20 μM etoposide (+) for 16 hours. Total cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. *refers to a non-specific band detected by the anti pS516Chk2, used as a control for Chk2 kinase activity. γH2AX and actin levels were used as controls for DSBs induction and equal loading, respectively. ( E ) Quantification of SUMO-HIC1 to total HIC1 (FLAG) for lanes 5 to 8 in panel D) was performed with the Fujifilm MultiGauge software.
Figure Legend Snippet: The increase of HIC1 SUMOylation upon irreparable DSB induction by etoposide requires ATM but not its effector kinase Chk2 ( A ) HEK293T cells were transfected with the FLAG and FLAG-HIC1 vectors. 48 hours after transfection, cells were pre-incubated or not with the Chk2 inhibitor (Chk2i) for 1 hour and then with etoposide for 16 hours as indicated. Cell extracts were prepared and Western blotting was performed with the indicated antibodies. *refers to non-specific bands detected by the anti pS516Chk2 (autophosphoylation) and by the anti pS20P53 (Chk2 target) antibodies. γH2AX and actin levels were used as controls for DSBs induction and equal loading, respectively. ( B ) To control for the inhibition of Chk2, HEK293T cells were transfected and pre-incubated or not with the Chk2 inhibitor (Chk2i) for 1 hour and then with etoposide for 16 hours exactly as in panel A) before lysis and Western blot analyses with the indicated antibodies ( C ) HEK293T cells grown in normal medium were transfected with siRNA control (siCtrl) or with a Chk2 siRNA pool (siChk2). Total RNAs were extracted and the mRNA expression levels of Chk2 were assessed by qRT-PCR. Values were normalized to 18S . ( D ) HEK293T cells were transfected either with non-target control siRNA (siCtrl) or with a Chk2 siRNA pool (siChk2) before being transfected with the indicated combination of FLAG, FLAG-HIC1 and SUMO-2 expression vectors. Cells were either incubated with DMSO (–) or with 20 μM etoposide (+) for 16 hours. Total cell extracts were prepared and analyzed by Western blotting with the indicated antibodies. *refers to a non-specific band detected by the anti pS516Chk2, used as a control for Chk2 kinase activity. γH2AX and actin levels were used as controls for DSBs induction and equal loading, respectively. ( E ) Quantification of SUMO-HIC1 to total HIC1 (FLAG) for lanes 5 to 8 in panel D) was performed with the Fujifilm MultiGauge software.

Techniques Used: Transfection, Incubation, Western Blot, Inhibition, Lysis, Expressing, Quantitative RT-PCR, Activity Assay, Software

Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter ( A ) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in IPH buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. ( B ) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P
Figure Legend Snippet: Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter ( A ) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in IPH buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. ( B ) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [ 6 ], as previously described [ 46 ]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P

Techniques Used: Transfection, Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Repairable DNA double-strand breaks (DSBs) induced by a 1 hour etoposide treatment do not lead to an ATM-dependent increase of HIC1 SUMOylation ( A ) Etoposide-induced non-repairable DSBs lead to an increase of HIC1 SUMOylation. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, SENP2 and SUMO2 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or DMSO (–) as control before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour or 16 hours. Cell extracts were prepared as described in panel A) and analyzed by immunoblotting using the indicated antibodies. Quantification of SUMO-HIC1 to total HIC1 (FLAG) was performed with the Fujifilm MultiGauge software (Bottom Panel) ( C ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour. Transfected cells were pre-treated or not with the following inhibitors (Wortmannin; ATMi, ATM inhibitor and DNAPKcsi, DNA-PKcs inhibitor) 1 hour before etoposide treatment, as indicated. Cell extracts were prepared as described in panel B) and analyzed by immunoblotting using the indicated antibodies. ( D ) HEK293T cells were co-transfected with the indicated combinations of expression vectors for FLAG-HIC1 and MTA1 and then incubated for 1 hour in etoposide or with DMSO as control. After lysis in IPH buffer, cells extracts were co-immunoprecipitated with anti-HIC1 antibodies. The immunoprecipitates as well as 2% of the whole cell extract (Input) were analyzed by Western blotting with the anti FLAG and anti MTA1 antibody. Note that the MTA1 antibodies detect a doublet of endogenous proteins in non transfected cells whereas the ectopically expressed MTA1 protein co-migrates with the upper band of the doublet (arrowheads).
Figure Legend Snippet: Repairable DNA double-strand breaks (DSBs) induced by a 1 hour etoposide treatment do not lead to an ATM-dependent increase of HIC1 SUMOylation ( A ) Etoposide-induced non-repairable DSBs lead to an increase of HIC1 SUMOylation. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, SENP2 and SUMO2 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or DMSO (–) as control before direct lysis in denaturing conditions. Total cell extracts were analyzed by Western Blotting (WB) using the indicated antibodies. ( B ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour or 16 hours. Cell extracts were prepared as described in panel A) and analyzed by immunoblotting using the indicated antibodies. Quantification of SUMO-HIC1 to total HIC1 (FLAG) was performed with the Fujifilm MultiGauge software (Bottom Panel) ( C ) HEK 293T cells were transfected with FLAG-HIC1 and treated with etoposide or DMSO for 1 hour. Transfected cells were pre-treated or not with the following inhibitors (Wortmannin; ATMi, ATM inhibitor and DNAPKcsi, DNA-PKcs inhibitor) 1 hour before etoposide treatment, as indicated. Cell extracts were prepared as described in panel B) and analyzed by immunoblotting using the indicated antibodies. ( D ) HEK293T cells were co-transfected with the indicated combinations of expression vectors for FLAG-HIC1 and MTA1 and then incubated for 1 hour in etoposide or with DMSO as control. After lysis in IPH buffer, cells extracts were co-immunoprecipitated with anti-HIC1 antibodies. The immunoprecipitates as well as 2% of the whole cell extract (Input) were analyzed by Western blotting with the anti FLAG and anti MTA1 antibody. Note that the MTA1 antibodies detect a doublet of endogenous proteins in non transfected cells whereas the ectopically expressed MTA1 protein co-migrates with the upper band of the doublet (arrowheads).

Techniques Used: Transfection, Expressing, Incubation, Lysis, Western Blot, Software, Immunoprecipitation

22) Product Images from "Etoposide Quinone Is a Covalent Poison of Human Topoisomerase IIβ"

Article Title: Etoposide Quinone Is a Covalent Poison of Human Topoisomerase IIβ

Journal: Biochemistry

doi: 10.1021/bi500421q

Etoposide quinone inhibits DNA ligation mediated by topoisomerase IIβ. DNA cleavage reactions were initiated in the absence (open circles, ND) or presence (closed circles, EQ) of 30 μM etoposide quinone. The DNA cleavage–ligation equilibrium was established at 37 °C, and ligation was initiated by cooling samples to 0 °C. The level of DNA cleavage observed at equilibrium for each reaction was set to 100% at time zero. Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Etoposide quinone inhibits DNA ligation mediated by topoisomerase IIβ. DNA cleavage reactions were initiated in the absence (open circles, ND) or presence (closed circles, EQ) of 30 μM etoposide quinone. The DNA cleavage–ligation equilibrium was established at 37 °C, and ligation was initiated by cooling samples to 0 °C. The level of DNA cleavage observed at equilibrium for each reaction was set to 100% at time zero. Error bars represent the standard deviation of three independent experiments.

Techniques Used: DNA Ligation, Ligation, Standard Deviation

Etoposide quinone does not require ATP to induce optimal DNA cleavage mediated by topoisomerase IIβ. DNA cleavage reactions of etoposide (left panel, Etop, red) or etoposide quinone (right panel, EQ, blue) were carried out in the absence (closed bars) or presence (open bars) of 0.25 mM ATP. Control reactions conducted in the absence of drug are shown (ND). Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Etoposide quinone does not require ATP to induce optimal DNA cleavage mediated by topoisomerase IIβ. DNA cleavage reactions of etoposide (left panel, Etop, red) or etoposide quinone (right panel, EQ, blue) were carried out in the absence (closed bars) or presence (open bars) of 0.25 mM ATP. Control reactions conducted in the absence of drug are shown (ND). Error bars represent the standard deviation of three independent experiments.

Techniques Used: Standard Deviation

Etoposide quinone enhances DNA cleavage mediated by human topoisomerase IIβ. DNA cleavage was carried out in the presence of etoposide (Etop, red) or etoposide quinone (EQ, blue) in the absence (closed circles) or presence (open circles) of 50 μM dithiothreitol (DTT). The left panel shows drug titrations, and the right panel shows a time course for DNA cleavage in the presence of 15 μM etoposide quinone. Error bars represent the standard deviation of three or more independent experiments.
Figure Legend Snippet: Etoposide quinone enhances DNA cleavage mediated by human topoisomerase IIβ. DNA cleavage was carried out in the presence of etoposide (Etop, red) or etoposide quinone (EQ, blue) in the absence (closed circles) or presence (open circles) of 50 μM dithiothreitol (DTT). The left panel shows drug titrations, and the right panel shows a time course for DNA cleavage in the presence of 15 μM etoposide quinone. Error bars represent the standard deviation of three or more independent experiments.

Techniques Used: Standard Deviation

Etoposide quinone inactivates human topoisomerase IIβ when incubated with the enzyme prior to the addition of DNA. The enzyme was incubated in the presence of 15 μM etoposide quinone (closed circles, blue) prior to a DNA cleavage assay. The inset shows cleavage levels established following incubation for 6 min in the absence of drug (ND, black bar) or in the presence of 30 μM etoposide (Etop, red bar), 15 μM etoposide quinone and DTT (EQ+DTT, open blue bar), or 15 μM etoposide quinone in the absence of DTT (EQ, blue bar). Error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Etoposide quinone inactivates human topoisomerase IIβ when incubated with the enzyme prior to the addition of DNA. The enzyme was incubated in the presence of 15 μM etoposide quinone (closed circles, blue) prior to a DNA cleavage assay. The inset shows cleavage levels established following incubation for 6 min in the absence of drug (ND, black bar) or in the presence of 30 μM etoposide (Etop, red bar), 15 μM etoposide quinone and DTT (EQ+DTT, open blue bar), or 15 μM etoposide quinone in the absence of DTT (EQ, blue bar). Error bars represent the standard deviation of three independent experiments.

Techniques Used: Incubation, DNA Cleavage Assay, Standard Deviation

Etoposide quinone is a covalent poison of topoisomerase IIβ. In the left panel, etoposide quinone enhancement of DNA cleavage is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence (blue bars) or presence (open blue bars) of DTT. Reaction mixtures contained no drug (ND) or 30 μM etoposide quinone in mixtures that included DTT at the time of DNA cleavage (Pre EQ) or DTT that was added (for an additional 6 min) after cleavage complexes were formed (Post EQ). In the right panel, etoposide quinone does not form DNA lesions that poison topoisomerase IIβ. DNA was incubated without (−EQ, open orange bars) or with (+EQ, orange bars) 30 μM etoposide quinone. DNA was purified from free drug and used in DNA cleavage reactions mediated by topoisomerase IIβ. DNA cleavage reactions were performed in the absence of drug (ND) or in the presence of 30 μM etoposide quinone (EQ). In all cases, error bars represent the standard deviation of three independent experiments.
Figure Legend Snippet: Etoposide quinone is a covalent poison of topoisomerase IIβ. In the left panel, etoposide quinone enhancement of DNA cleavage is not reversed by the addition of reducing agents after DNA cleavage complexes have been established. DNA cleavage reactions were carried out in the absence (blue bars) or presence (open blue bars) of DTT. Reaction mixtures contained no drug (ND) or 30 μM etoposide quinone in mixtures that included DTT at the time of DNA cleavage (Pre EQ) or DTT that was added (for an additional 6 min) after cleavage complexes were formed (Post EQ). In the right panel, etoposide quinone does not form DNA lesions that poison topoisomerase IIβ. DNA was incubated without (−EQ, open orange bars) or with (+EQ, orange bars) 30 μM etoposide quinone. DNA was purified from free drug and used in DNA cleavage reactions mediated by topoisomerase IIβ. DNA cleavage reactions were performed in the absence of drug (ND) or in the presence of 30 μM etoposide quinone (EQ). In all cases, error bars represent the standard deviation of three independent experiments.

Techniques Used: Incubation, Purification, Standard Deviation

Etoposide quinone induces DNA cleavage via an enzyme-mediated mechanism. Control reactions were conducted in the absence of enzyme or drug (DNA Control), in the presence of 30 μM etoposide quinone without enzyme (+EQ −hTIIβ), or in the presence of topoisomerase IIβ without drug (−EQ +hTIIβ). All other reaction mixtures contained topoisomerase IIβ and 30 μM etoposide quinone. DNA cleavage reactions were terminated by the addition of SDS (+EQ +hTIIβ). To determine whether cleaved DNA was protein-linked, proteinase K treatment was omitted (−ProK). The reversibility of DNA cleavage was examined by adding EDTA (EDTA) or 0.5 M salt (NaCl) prior to SDS. The level of enzyme-mediated DNA cleavage in the absence of etoposide quinone was set to 1 in the bottom panel, and all other reactions were expressed relative to that value. Error bars represent standard deviations for three independent experiments. A representative agarose gel stained with ethidium bromide is shown at the top. The positions of supercoiled (form I, FI), nicked circular (form II, FII), and linear (form III, FIII) molecules are indicated at the left.
Figure Legend Snippet: Etoposide quinone induces DNA cleavage via an enzyme-mediated mechanism. Control reactions were conducted in the absence of enzyme or drug (DNA Control), in the presence of 30 μM etoposide quinone without enzyme (+EQ −hTIIβ), or in the presence of topoisomerase IIβ without drug (−EQ +hTIIβ). All other reaction mixtures contained topoisomerase IIβ and 30 μM etoposide quinone. DNA cleavage reactions were terminated by the addition of SDS (+EQ +hTIIβ). To determine whether cleaved DNA was protein-linked, proteinase K treatment was omitted (−ProK). The reversibility of DNA cleavage was examined by adding EDTA (EDTA) or 0.5 M salt (NaCl) prior to SDS. The level of enzyme-mediated DNA cleavage in the absence of etoposide quinone was set to 1 in the bottom panel, and all other reactions were expressed relative to that value. Error bars represent standard deviations for three independent experiments. A representative agarose gel stained with ethidium bromide is shown at the top. The positions of supercoiled (form I, FI), nicked circular (form II, FII), and linear (form III, FIII) molecules are indicated at the left.

Techniques Used: Agarose Gel Electrophoresis, Staining

Etoposide quinone induces a high ratio of double-stranded DNA breaks (DSB) to single-stranded DNA breaks (SSB). DNA strand breaks generated by human topoisomerase IIβ were monitored in reaction mixtures containing no drug (ND, black), 30 μM etoposide (Etop, red), or 15 μM etoposide quinone (EQ, blue). Double- and single-stranded DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to linear and nicked molecules, respectively. Error bars represent the standard deviation of three independent experiments. Results of an unpaired two-tailed t test are shown (** p = 0.001).
Figure Legend Snippet: Etoposide quinone induces a high ratio of double-stranded DNA breaks (DSB) to single-stranded DNA breaks (SSB). DNA strand breaks generated by human topoisomerase IIβ were monitored in reaction mixtures containing no drug (ND, black), 30 μM etoposide (Etop, red), or 15 μM etoposide quinone (EQ, blue). Double- and single-stranded DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to linear and nicked molecules, respectively. Error bars represent the standard deviation of three independent experiments. Results of an unpaired two-tailed t test are shown (** p = 0.001).

Techniques Used: Generated, Plasmid Preparation, Standard Deviation, Two Tailed Test

23) Product Images from "Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality"

Article Title: Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality

Journal: Stem Cells Translational Medicine

doi: 10.1002/sctm.17-0116

Human induced pluripotent stem cell (hiPSC) clones exhibiting the highest degree of etoposide sensitivity also demonstrated good hiPSC clonal morphology and pluripotent gene expression patterns consistent with pluripotent control cell lines. (A): ×40 images of hiPSC clones. Red outline highlights clones (RT4.1, RT6.21) displaying the best morphology (clean, distinct borders). (B): Immunofluorescence detection (×40) of SSEA‐3 and TRA‐1–60 expression in hiPSCs. (C): qPCR of RNA isolated from five of our hiPSC lines (RT), two control hiPSC lines (IMR90, iPSf2) and two control ESC lines (H9, H13). Red outline highlights the two clones (RT4.1, RT6.21) exhibiting pluripotent gene expression patterns most similar to those displayed by control pluripotent cells. (D): RT4.1 and RT6.21 clones successfully formed teratomas in athymic nude mice. Scan bars = 50 µM. (E): Annexin V/PI staining of hiPSC cells treated w/wo etoposide for 24 hours and plotted as a percent of DMSO control. Red outline highlights two hiPSC lines (RT4.1, 6.21) that demonstrated the greatest sensitivity to etoposide. Data used to calculate the individual means was generated from a minimum of five biological replications. Error bars represent the SD calculated around an individual mean. Abbreviation: DMSO, dimethyl sulfoxide.
Figure Legend Snippet: Human induced pluripotent stem cell (hiPSC) clones exhibiting the highest degree of etoposide sensitivity also demonstrated good hiPSC clonal morphology and pluripotent gene expression patterns consistent with pluripotent control cell lines. (A): ×40 images of hiPSC clones. Red outline highlights clones (RT4.1, RT6.21) displaying the best morphology (clean, distinct borders). (B): Immunofluorescence detection (×40) of SSEA‐3 and TRA‐1–60 expression in hiPSCs. (C): qPCR of RNA isolated from five of our hiPSC lines (RT), two control hiPSC lines (IMR90, iPSf2) and two control ESC lines (H9, H13). Red outline highlights the two clones (RT4.1, RT6.21) exhibiting pluripotent gene expression patterns most similar to those displayed by control pluripotent cells. (D): RT4.1 and RT6.21 clones successfully formed teratomas in athymic nude mice. Scan bars = 50 µM. (E): Annexin V/PI staining of hiPSC cells treated w/wo etoposide for 24 hours and plotted as a percent of DMSO control. Red outline highlights two hiPSC lines (RT4.1, 6.21) that demonstrated the greatest sensitivity to etoposide. Data used to calculate the individual means was generated from a minimum of five biological replications. Error bars represent the SD calculated around an individual mean. Abbreviation: DMSO, dimethyl sulfoxide.

Techniques Used: Expressing, Clone Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Isolation, Mouse Assay, Staining, Generated

Poor human induced pluripotent stem cell (hiPSC) clone maintenance and cellular differentiation result in a rapid loss of etoposide sensitivity. (A, B): hiPSC cultures in which spontaneously differentiated cells were removed via MC exhibited increased etoposide sensitivity (B) compared to the same cultures prior to MC (A). (C): Terminally differentiated human fibroblast cells demonstrated no detectable apoptosis or cell death following treatment with etoposide. Data points were derived from the average of ten unique primary human fibroblast cultures and plotted as a percentage of DMSO control. (D): hiPSCs undergoing directed cardiac differentiation display a progressive decrease in etoposide sensitivity. All data points were derived from the average of three technical replicate samples stained with Annexin V/PI, and normalized to DMSO treated cells. Error bars represent the standard deviation calculated around an individual mean. Abbreviations: DMSO, dimethyl sulfoxide; MC, mechanical cleaning.
Figure Legend Snippet: Poor human induced pluripotent stem cell (hiPSC) clone maintenance and cellular differentiation result in a rapid loss of etoposide sensitivity. (A, B): hiPSC cultures in which spontaneously differentiated cells were removed via MC exhibited increased etoposide sensitivity (B) compared to the same cultures prior to MC (A). (C): Terminally differentiated human fibroblast cells demonstrated no detectable apoptosis or cell death following treatment with etoposide. Data points were derived from the average of ten unique primary human fibroblast cultures and plotted as a percentage of DMSO control. (D): hiPSCs undergoing directed cardiac differentiation display a progressive decrease in etoposide sensitivity. All data points were derived from the average of three technical replicate samples stained with Annexin V/PI, and normalized to DMSO treated cells. Error bars represent the standard deviation calculated around an individual mean. Abbreviations: DMSO, dimethyl sulfoxide; MC, mechanical cleaning.

Techniques Used: Cell Differentiation, Derivative Assay, Staining, Standard Deviation

Analysis of 115 unique human induced pluripotent stem cell (hiPSC) clones by etoposide sensitivity assay (ESA); ESA, and not flow‐based TRA‐1–60/SSEA‐4 staining, was able to decipher a significant difference between hiPSC clones originally produced from Sendai or Lentiviral reprogramming methods. (A): Example SSEA‐4/TRA‐1–60 dot plot including Tra+ SS+ values used to determine percent SSEA‐4/TRA‐1–60 expression. (B, C): Mean dual (+) SSEA‐4/TRA‐1–60 value(s) calculated for hiPSC clones (B) combined ( n = 100) and (C) separately by lentivirus ( n = 57) or Sendai virus ( n = 43). (D): A summary graph incorporating 115 hiPSC clones treated with etoposide (or DMSO control) for 24 hours. and subsequently analyzed by ESA (Annexin V/PI). Red dots represent the median value of the associated treatment. (E, F): Mean EC50 value(s) calculated for hiPSC clones (E) regardless of reprogramming strategy ( n = 115) and (F) separately by lentivirus ( n = 60) or Sendai virus ( n = 55). Mean passage numbers for lenti‐ and Sendai‐reprogrammed hiPSC clones were 9.56 ± 2.14 and 8.27 ± 2.21, respectively, with error calculations representing the standard deviation around an individual mean.
Figure Legend Snippet: Analysis of 115 unique human induced pluripotent stem cell (hiPSC) clones by etoposide sensitivity assay (ESA); ESA, and not flow‐based TRA‐1–60/SSEA‐4 staining, was able to decipher a significant difference between hiPSC clones originally produced from Sendai or Lentiviral reprogramming methods. (A): Example SSEA‐4/TRA‐1–60 dot plot including Tra+ SS+ values used to determine percent SSEA‐4/TRA‐1–60 expression. (B, C): Mean dual (+) SSEA‐4/TRA‐1–60 value(s) calculated for hiPSC clones (B) combined ( n = 100) and (C) separately by lentivirus ( n = 57) or Sendai virus ( n = 43). (D): A summary graph incorporating 115 hiPSC clones treated with etoposide (or DMSO control) for 24 hours. and subsequently analyzed by ESA (Annexin V/PI). Red dots represent the median value of the associated treatment. (E, F): Mean EC50 value(s) calculated for hiPSC clones (E) regardless of reprogramming strategy ( n = 115) and (F) separately by lentivirus ( n = 60) or Sendai virus ( n = 55). Mean passage numbers for lenti‐ and Sendai‐reprogrammed hiPSC clones were 9.56 ± 2.14 and 8.27 ± 2.21, respectively, with error calculations representing the standard deviation around an individual mean.

Techniques Used: Sensitive Assay, Flow Cytometry, Staining, Clone Assay, Produced, Expressing, Standard Deviation

Large scale ESA revealed a positive correlation between ESA derived EC50 values and pluripotent gene expression. (A): Heat map (yellow = RNA expression) of 89 human induced pluripotent stem cell (hiPSC) clones analyzed for seven common markers of pluripotency. Lenti‐ or Sendai virus reprogramming strategy for each clone is indicated by a corresponding orange (lentivirus) or blue (Sendai) dots appearing below heat map. (B): PCA of SSEA‐4/TRA‐1‐60 values from hiPSCs created with lentivirus ( n = 18) or Sendai virus ( n = 18) compared with qPCR Ct values generated from expression of the seven pluripotency‐related genes listed in Figure 3A. Sphere size is relatively proportional to percent SSEA‐4/TRA‐1‐60 values (i.e., large spheres equate to a high level of SSEA‐4/TRA‐1‐60 coexpression). (C): PCA of ESA EC50 values from hiPSC clones created with lentivirus ( n = 60) or Sendai virus ( n = 29) compared with Ct values as described in Figure 5B. Sphere size is relatively proportional to EC50 values (i.e., large spheres equate to high EC50 values). Abbreviation: ESA, etoposide sensitivity assay.
Figure Legend Snippet: Large scale ESA revealed a positive correlation between ESA derived EC50 values and pluripotent gene expression. (A): Heat map (yellow = RNA expression) of 89 human induced pluripotent stem cell (hiPSC) clones analyzed for seven common markers of pluripotency. Lenti‐ or Sendai virus reprogramming strategy for each clone is indicated by a corresponding orange (lentivirus) or blue (Sendai) dots appearing below heat map. (B): PCA of SSEA‐4/TRA‐1‐60 values from hiPSCs created with lentivirus ( n = 18) or Sendai virus ( n = 18) compared with qPCR Ct values generated from expression of the seven pluripotency‐related genes listed in Figure 3A. Sphere size is relatively proportional to percent SSEA‐4/TRA‐1‐60 values (i.e., large spheres equate to a high level of SSEA‐4/TRA‐1‐60 coexpression). (C): PCA of ESA EC50 values from hiPSC clones created with lentivirus ( n = 60) or Sendai virus ( n = 29) compared with Ct values as described in Figure 5B. Sphere size is relatively proportional to EC50 values (i.e., large spheres equate to high EC50 values). Abbreviation: ESA, etoposide sensitivity assay.

Techniques Used: Derivative Assay, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Generated, Clone Assay, Sensitive Assay

A maximum EC50 cutoff value of 300 nM quantitatively identifies good quality human induced pluripotent stem cell (hiPSC) clones. (A): EC50 maximum cutoff plot generated from the principal components analysis‐based examination of hiPSC gene expression profiles and etoposide sensitivity EC50 data. Outliers are defined as hiPSC clones exhibiting EC50 values greater than 300 nM. Abbreviation: EC, effective concentration.
Figure Legend Snippet: A maximum EC50 cutoff value of 300 nM quantitatively identifies good quality human induced pluripotent stem cell (hiPSC) clones. (A): EC50 maximum cutoff plot generated from the principal components analysis‐based examination of hiPSC gene expression profiles and etoposide sensitivity EC50 data. Outliers are defined as hiPSC clones exhibiting EC50 values greater than 300 nM. Abbreviation: EC, effective concentration.

Techniques Used: Generated, Expressing, Clone Assay, Concentration Assay

24) Product Images from "Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells"

Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

Journal: Molecular Pharmacology

doi: 10.1124/mol.109.054999

Etoposide triggers caspase-3-dependent apoptosis in SK-N-AS cells. A, cells were treated with 50 μM etoposide for 48 h and harvested. The cytosolic fractions were obtained by a digitonin-based subcellular fractionation procedure. One hundred micrograms of cytosolic (Cy) and mitochondrial protein fractions (Mi) was analyzed by SDS-polyacrylamide gel electrophoresis, and cytochrome c and β-actin levels were determined by immunoblotting. B, immunoblot analysis of caspase-9 and β-actin in cell lysates treated with 50 μM etoposide for 48 h. C, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-9 inhibitor (z-LEHD-fmk) for 48 h. D, immunoblot analysis of PKCδ and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. E, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. F, cells were treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide triggers caspase-3-dependent apoptosis in SK-N-AS cells. A, cells were treated with 50 μM etoposide for 48 h and harvested. The cytosolic fractions were obtained by a digitonin-based subcellular fractionation procedure. One hundred micrograms of cytosolic (Cy) and mitochondrial protein fractions (Mi) was analyzed by SDS-polyacrylamide gel electrophoresis, and cytochrome c and β-actin levels were determined by immunoblotting. B, immunoblot analysis of caspase-9 and β-actin in cell lysates treated with 50 μM etoposide for 48 h. C, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-9 inhibitor (z-LEHD-fmk) for 48 h. D, immunoblot analysis of PKCδ and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. E, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. F, cells were treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Fractionation, Polyacrylamide Gel Electrophoresis, Staining

Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.
Figure Legend Snippet: Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.

Techniques Used: Activation Assay, Expressing

Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Transfection, Staining

Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Activation Assay, Transfection, Staining

Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.
Figure Legend Snippet: Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.

Techniques Used: Transfection, Staining

Etoposide induces PKCδ-mediated apoptosis in SK-N-AS cells. A, immunoblot analysis of PKCδ and β-actin in cell lysates after the treatment with 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were treated as described above, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements. C, immunoblot analysis of PKCδ and β-actin from whole-cell lysates after the transfection with 100 nM nontargeting siRNA (SC) or PKCδ -specific siRNA for 72 h (inset), cells were treated with 50 μM etoposide for an additional 48 h, and apoptosis was determined by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were treated with 10 nM Gö6976 for 2 h followed by 50 μM etoposide for 48 h, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements.
Figure Legend Snippet: Etoposide induces PKCδ-mediated apoptosis in SK-N-AS cells. A, immunoblot analysis of PKCδ and β-actin in cell lysates after the treatment with 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were treated as described above, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements. C, immunoblot analysis of PKCδ and β-actin from whole-cell lysates after the transfection with 100 nM nontargeting siRNA (SC) or PKCδ -specific siRNA for 72 h (inset), cells were treated with 50 μM etoposide for an additional 48 h, and apoptosis was determined by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were treated with 10 nM Gö6976 for 2 h followed by 50 μM etoposide for 48 h, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements.

Techniques Used: Staining, Flow Cytometry, Cytometry, Transfection

Etoposide inhibits the proliferation and triggers apoptosis in SK-N-AS cells. A, cells were treated with 10, 25, 50, or 100 μM etoposide for 48 h, and cell proliferation was determined by using the CellTiter 96 Aqueous One solution cell proliferation assay reagent. Values are representative of three independent experiments, and error bars show S.D. from triplicate counts. B, cells were treated with 50 μM etoposide for 48 h, harvested, and apoptosis was determined by FACScan analysis as described under Materials and Methods . Compensation was executed for each experiment using untreated cells stained with Annexin V and propidium iodide. Error bars show S.D. from triplicate measurements. C, fluorescence microscopy images at 40× magnification of Hoechst 33342-stained nuclei of untreated cells or cells treated with 50 μM etoposide for 48 h. D, quantification of apoptosis was carried out by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide inhibits the proliferation and triggers apoptosis in SK-N-AS cells. A, cells were treated with 10, 25, 50, or 100 μM etoposide for 48 h, and cell proliferation was determined by using the CellTiter 96 Aqueous One solution cell proliferation assay reagent. Values are representative of three independent experiments, and error bars show S.D. from triplicate counts. B, cells were treated with 50 μM etoposide for 48 h, harvested, and apoptosis was determined by FACScan analysis as described under Materials and Methods . Compensation was executed for each experiment using untreated cells stained with Annexin V and propidium iodide. Error bars show S.D. from triplicate measurements. C, fluorescence microscopy images at 40× magnification of Hoechst 33342-stained nuclei of untreated cells or cells treated with 50 μM etoposide for 48 h. D, quantification of apoptosis was carried out by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Proliferation Assay, Staining, Fluorescence, Microscopy

25) Product Images from "HSP27 Is a Ubiquitin-Binding Protein Involved in I-?B? Proteasomal Degradation"

Article Title: HSP27 Is a Ubiquitin-Binding Protein Involved in I-?B? Proteasomal Degradation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.16.5790-5802.2003

HSP27 enhances I-κBα degradation by the proteasome. (A) Control-transfected and HSP27-transfected U937 cells were treated as indicated (VP16, 100 μM, 4 h; ΤNF-α, 20 ng/ml, 4 h; MG132, 25 μM) before monitoring of I-κBα expression by Western blotting. HSC70 served as a loading control. (B) Control-transfected (white bars) and HSP27-transfected (black bars) U937 cells were treated with etoposide (100 μM) for the indicated times before measurement of I-κBα cellular content by flow cytometry (MFI, mean fluorescence index). (C) REG cells were transfected with an empty plasmid (Control) or a plasmid encoding wild-type HSP27 or the indicated deletion mutants of HSP27 and then were treated with etoposide (VP16, 100 μM, 24 h) before measurement of I-κBα cellular content by flow cytometry as described for panel B. Results are the means and standard deviations for four independent experiments.
Figure Legend Snippet: HSP27 enhances I-κBα degradation by the proteasome. (A) Control-transfected and HSP27-transfected U937 cells were treated as indicated (VP16, 100 μM, 4 h; ΤNF-α, 20 ng/ml, 4 h; MG132, 25 μM) before monitoring of I-κBα expression by Western blotting. HSC70 served as a loading control. (B) Control-transfected (white bars) and HSP27-transfected (black bars) U937 cells were treated with etoposide (100 μM) for the indicated times before measurement of I-κBα cellular content by flow cytometry (MFI, mean fluorescence index). (C) REG cells were transfected with an empty plasmid (Control) or a plasmid encoding wild-type HSP27 or the indicated deletion mutants of HSP27 and then were treated with etoposide (VP16, 100 μM, 24 h) before measurement of I-κBα cellular content by flow cytometry as described for panel B. Results are the means and standard deviations for four independent experiments.

Techniques Used: Transfection, Expressing, Western Blot, Flow Cytometry, Cytometry, Fluorescence, Plasmid Preparation

HSP27 increases nuclear NF-κB content, DNA binding, and transcriptional activity in stressed cells. (A) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), or IL-1β (1 ng/ml). The level of nuclear NF-κB expression was determined by flow cytometry (MFI, mean fluorescence index). (B) Nuclear extracts from control-transfected and HSP27-transfected U937 cells either were left untreated or were treated for 4 h with etoposide (VP16, 100 μM) or ΤNF-α (20 ng/ml) and then were subjected to EMSAs with an NF-κB probe. As a control for binding specificity, the extracts were preincubated with a 50-fold molar excess of unlabeled oligonucleotide (Unl.). Supershift analysis was performed by preincubation of the extracts with an anti-p50 or an anti-p65 polyclonal antibody before EMSAs. (C) Control-transfected (white bars) and HSP27-overexpressing (black bars) cells were transiently transfected with an NF-κB luciferase reporter plasmid and then either were left untreated or were treated for 4 h (U937 cells) or 10 h (MEF cells) with 100 μM VP16 or 20 ng of ΤNF-α/ml. Cotransfection of a thymidine kinase- Renilla luciferase plasmid was used to normalize for transfection efficiency. AU, arbitrary units. Results are the means and standard deviations for three independent experiments.
Figure Legend Snippet: HSP27 increases nuclear NF-κB content, DNA binding, and transcriptional activity in stressed cells. (A) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), or IL-1β (1 ng/ml). The level of nuclear NF-κB expression was determined by flow cytometry (MFI, mean fluorescence index). (B) Nuclear extracts from control-transfected and HSP27-transfected U937 cells either were left untreated or were treated for 4 h with etoposide (VP16, 100 μM) or ΤNF-α (20 ng/ml) and then were subjected to EMSAs with an NF-κB probe. As a control for binding specificity, the extracts were preincubated with a 50-fold molar excess of unlabeled oligonucleotide (Unl.). Supershift analysis was performed by preincubation of the extracts with an anti-p50 or an anti-p65 polyclonal antibody before EMSAs. (C) Control-transfected (white bars) and HSP27-overexpressing (black bars) cells were transiently transfected with an NF-κB luciferase reporter plasmid and then either were left untreated or were treated for 4 h (U937 cells) or 10 h (MEF cells) with 100 μM VP16 or 20 ng of ΤNF-α/ml. Cotransfection of a thymidine kinase- Renilla luciferase plasmid was used to normalize for transfection efficiency. AU, arbitrary units. Results are the means and standard deviations for three independent experiments.

Techniques Used: Binding Assay, Activity Assay, Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence, Luciferase, Plasmid Preparation, Cotransfection

HSP27 enhances the degradation of ubiquitinated proteins in stressed cells. (A) Control-transfected (Co) and HSP27-transfected U937 cells either were left untreated or were treated with 100 μM etoposide (VP16) for 4 h in the absence or presence of 25 μM MG132. Protein ubiquitination (Ub-proteins) was monitored by Western blotting with an antiubiquitin antibody. α-actin served as the loading control. (B) Densitometry analysis of Ub-proteins in three independent experiments (mean and standard deviation) similar to that shown in panel A. AU, arbitrary units. (C) U937 cells either were left untreated (NT) or were exposed for 1 h to 42°C (HS) and then were incubated at 37°C for the indicated times. The indicated proteins were studied by Western blotting. α-Actin served as the loading control. (D) Densitometry analysis of Ub-proteins shown in panel C.
Figure Legend Snippet: HSP27 enhances the degradation of ubiquitinated proteins in stressed cells. (A) Control-transfected (Co) and HSP27-transfected U937 cells either were left untreated or were treated with 100 μM etoposide (VP16) for 4 h in the absence or presence of 25 μM MG132. Protein ubiquitination (Ub-proteins) was monitored by Western blotting with an antiubiquitin antibody. α-actin served as the loading control. (B) Densitometry analysis of Ub-proteins in three independent experiments (mean and standard deviation) similar to that shown in panel A. AU, arbitrary units. (C) U937 cells either were left untreated (NT) or were exposed for 1 h to 42°C (HS) and then were incubated at 37°C for the indicated times. The indicated proteins were studied by Western blotting. α-Actin served as the loading control. (D) Densitometry analysis of Ub-proteins shown in panel C.

Techniques Used: Transfection, Western Blot, Standard Deviation, Incubation

HSP27, phosphorylated I-κBα, and PA700 can be found in the same cellular protein fraction. (A) Lysates from HSP27-transfected U937 cells not treated (triangles) or treated (squares) with etoposide (100 μM, 4 h) were centrifuged and fractionated with a Superose-6 column. Fractions were tested for hydrolysis of the substrate Suc-LLVY-AMC. AU, arbitrary units. (B) The presence of PA700, HSP27, and phosphorylated I-κBα (P-I-κBα) in fractions of cell extracts from nontreated (NT) and etoposide-treated (VP16) cells in the presence of MG132 (25 μM) to stabilize phosphorylated I-κBα was determined by Western blotting. (C) Immunodetection of PA700 and P-I-κBα after immunoprecipitation (IP) of HSP27 in fraction 17 of cells exposed to etoposide in the presence of MG132. ch., chain.
Figure Legend Snippet: HSP27, phosphorylated I-κBα, and PA700 can be found in the same cellular protein fraction. (A) Lysates from HSP27-transfected U937 cells not treated (triangles) or treated (squares) with etoposide (100 μM, 4 h) were centrifuged and fractionated with a Superose-6 column. Fractions were tested for hydrolysis of the substrate Suc-LLVY-AMC. AU, arbitrary units. (B) The presence of PA700, HSP27, and phosphorylated I-κBα (P-I-κBα) in fractions of cell extracts from nontreated (NT) and etoposide-treated (VP16) cells in the presence of MG132 (25 μM) to stabilize phosphorylated I-κBα was determined by Western blotting. (C) Immunodetection of PA700 and P-I-κBα after immunoprecipitation (IP) of HSP27 in fraction 17 of cells exposed to etoposide in the presence of MG132. ch., chain.

Techniques Used: Transfection, Western Blot, Immunodetection, Immunoprecipitation

HSP27 enhances proteasome activation while inhibiting apoptosis. (A) U937 cells either were left untreated or were treated for 4 h with 100 μM etoposide (VP16) in the absence or presence of acetyl-calpastatin (25 μM), MG132 (25 μM), or lactacystin (25 μM) before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC (black bars; AU, arbitrary units) and the percentage of apoptotic cells after nuclear chromatin staining with Hoechst 33342 (gray bars). (B) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with 100 μM etoposide (VP16) or 20 ng of ΤNF-α/ml in the absence or presence of 25 μM MG132 before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC. Results are expressed as percentages (100% is the activity in VP16-treated, HSP27-transfected cells). Insets show Western blot analyses of HSP27 expression in control- and HSP27-transfected cells. (C) Kinetic analysis of Suc-LLVY-AMC cleavage activity (black bars) and apoptosis induction (gray bars) in control- and HSP27-transfected U937 cells treated with 100 μM etoposide for the indicated times. (D and E) Cells were treated with etoposide (100 μM, 4 h) in the presence of decreasing concentrations of MG132 or lactacystin, as indicated. Then, chromatin staining with Hoechst 33342 was used to measure the percentage of control-transfected and HSP27-transfected U937 apoptotic cells (D) or the percentage of cell survival induced by HSP27 overexpression (E). The percentage of proteasome inhibition (Suc-LLVY-AMC cleavage) is also indicated (E). Data are the means and standard deviations for three independent experiments.
Figure Legend Snippet: HSP27 enhances proteasome activation while inhibiting apoptosis. (A) U937 cells either were left untreated or were treated for 4 h with 100 μM etoposide (VP16) in the absence or presence of acetyl-calpastatin (25 μM), MG132 (25 μM), or lactacystin (25 μM) before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC (black bars; AU, arbitrary units) and the percentage of apoptotic cells after nuclear chromatin staining with Hoechst 33342 (gray bars). (B) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with 100 μM etoposide (VP16) or 20 ng of ΤNF-α/ml in the absence or presence of 25 μM MG132 before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC. Results are expressed as percentages (100% is the activity in VP16-treated, HSP27-transfected cells). Insets show Western blot analyses of HSP27 expression in control- and HSP27-transfected cells. (C) Kinetic analysis of Suc-LLVY-AMC cleavage activity (black bars) and apoptosis induction (gray bars) in control- and HSP27-transfected U937 cells treated with 100 μM etoposide for the indicated times. (D and E) Cells were treated with etoposide (100 μM, 4 h) in the presence of decreasing concentrations of MG132 or lactacystin, as indicated. Then, chromatin staining with Hoechst 33342 was used to measure the percentage of control-transfected and HSP27-transfected U937 apoptotic cells (D) or the percentage of cell survival induced by HSP27 overexpression (E). The percentage of proteasome inhibition (Suc-LLVY-AMC cleavage) is also indicated (E). Data are the means and standard deviations for three independent experiments.

Techniques Used: Activation Assay, Staining, Transfection, Activity Assay, Western Blot, Expressing, Over Expression, Inhibition

HSP27 associates with ubiquitin. (A) Cytoplasmic extracts obtained from control-transfected and HSP27-transfected U937 cells, either left untreated or treated with 100 μM etoposide (VP16) for 4 h, were incubated with a ubiquitin-agarose matrix. The presence of the indicated proteins in the input material was analyzed by Western blotting (left panels). Pull-down analysis of ubiquitin-agarose was performed in the absence or presence of 50 μg of free ubiquitin (free-Ub) before elution of bound proteins and Western blot analysis of HSPs (right panels). One representative experiment of three independent experiments is shown. (B) Western blot analysis of GST-monoubiquitin (GST-Ub) and GST-polyubiquitin (GST-polyUb) obtained in vitro as described in Material and Methods. (C) Cytosolic extracts from HSP27-overexpressing cells were incubated with GST alone, GST-Ub, or GST-polyUb. The presence of HSP27 in input material was checked by Western blotting after pull-down analysis and elution of bound proteins. One representative experiment of three experiments is shown.
Figure Legend Snippet: HSP27 associates with ubiquitin. (A) Cytoplasmic extracts obtained from control-transfected and HSP27-transfected U937 cells, either left untreated or treated with 100 μM etoposide (VP16) for 4 h, were incubated with a ubiquitin-agarose matrix. The presence of the indicated proteins in the input material was analyzed by Western blotting (left panels). Pull-down analysis of ubiquitin-agarose was performed in the absence or presence of 50 μg of free ubiquitin (free-Ub) before elution of bound proteins and Western blot analysis of HSPs (right panels). One representative experiment of three independent experiments is shown. (B) Western blot analysis of GST-monoubiquitin (GST-Ub) and GST-polyubiquitin (GST-polyUb) obtained in vitro as described in Material and Methods. (C) Cytosolic extracts from HSP27-overexpressing cells were incubated with GST alone, GST-Ub, or GST-polyUb. The presence of HSP27 in input material was checked by Western blotting after pull-down analysis and elution of bound proteins. One representative experiment of three experiments is shown.

Techniques Used: Transfection, Incubation, Western Blot, In Vitro

HSP27 associates with phosphorylated I-κBα. (A) Control-transfected and HSP27-transfected U937 cells were treated as indicated (VP16, 100 μM, 4 h; ΤNF-α, 20 ng/ml, 4 h; MG132, 25 μM) before monitoring of phosphorylated I-κBα (P-I-κBα) expression by Western blotting. HSC70 served as a loading control. (B) Total cell lysates were prepared from HSP27-transfected U937 cells either left untreated or treated for 4 h with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), and/or MG132 (25 μM) and then immunoprecipitated (IP) with the indicated antibodies. The indicated proteins were detected by Western blotting. (C) Control-transfected and HSP27-transfected U937 cells and control-transfected and HSP27-transfected MEF cells were transiently transfected with an empty vector or a plasmid containing a nonphosphorylatable, nondegradable mutant form of I-κBα (I-κBα S32A/S36A) (transfection efficiency measured with a β-galactosidase-expressing plasmid, 35 to 40%). At 36 h later, cells were treated with 100 μM etoposide (VP16) or 20 ng of TNF-α/ml for 4 h (U937 cells) or 24 h (MEF cells) before measurement of the percentage of apoptotic cells. Error bars indicate standard deviations ( n = 4).
Figure Legend Snippet: HSP27 associates with phosphorylated I-κBα. (A) Control-transfected and HSP27-transfected U937 cells were treated as indicated (VP16, 100 μM, 4 h; ΤNF-α, 20 ng/ml, 4 h; MG132, 25 μM) before monitoring of phosphorylated I-κBα (P-I-κBα) expression by Western blotting. HSC70 served as a loading control. (B) Total cell lysates were prepared from HSP27-transfected U937 cells either left untreated or treated for 4 h with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), and/or MG132 (25 μM) and then immunoprecipitated (IP) with the indicated antibodies. The indicated proteins were detected by Western blotting. (C) Control-transfected and HSP27-transfected U937 cells and control-transfected and HSP27-transfected MEF cells were transiently transfected with an empty vector or a plasmid containing a nonphosphorylatable, nondegradable mutant form of I-κBα (I-κBα S32A/S36A) (transfection efficiency measured with a β-galactosidase-expressing plasmid, 35 to 40%). At 36 h later, cells were treated with 100 μM etoposide (VP16) or 20 ng of TNF-α/ml for 4 h (U937 cells) or 24 h (MEF cells) before measurement of the percentage of apoptotic cells. Error bars indicate standard deviations ( n = 4).

Techniques Used: Transfection, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation, Mutagenesis

26) Product Images from "Hypoxia-Induced Cytotoxic Drug Resistance in Osteosarcoma Is Independent of HIF-1Alpha"

Article Title: Hypoxia-Induced Cytotoxic Drug Resistance in Osteosarcoma Is Independent of HIF-1Alpha

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065304

Cobalt Chloride stabilises and transcriptionally activates HIF-1α in normoxia but does not induce resistance. B, 24 hours after plating osteosarcoma cells were treated with cobalt chloride (791T 50 µM; HOS 25 µM; U2OS 25 µM) for 24 hours before treatment with a range of concentrations of cisplatin (791T 0–50 µM; HOS 0–25 µM; U2OS 0–200 µM), doxorubicin (791T 0–16 µM; HOS 0–5 µM; U2OS 0–40 µM) or etoposide (791T 0–50 µM; HOS 0–50 µM; U2OS 0–1000 µM). Following a one hour drug exposure cells were incubated with or without cobalt chloride for a further 72 hours before fixing and performing a sulphorhodamine-B assay. Graphs show the mean absorbance relative to the untreated controls (UnT) against the log of the drug concentrations and are the average of 3 independent experiments ± SEM. A, Whole cell lysates of cells treated with the above doses of cobalt chloride for the length of the experiment (96 hours) were harvested for western blotting to determine HIF-1α stabilisation and expression of downstream target CA IX. The western blots are representative of 3 independent experiments with GAPDH as a loading control.
Figure Legend Snippet: Cobalt Chloride stabilises and transcriptionally activates HIF-1α in normoxia but does not induce resistance. B, 24 hours after plating osteosarcoma cells were treated with cobalt chloride (791T 50 µM; HOS 25 µM; U2OS 25 µM) for 24 hours before treatment with a range of concentrations of cisplatin (791T 0–50 µM; HOS 0–25 µM; U2OS 0–200 µM), doxorubicin (791T 0–16 µM; HOS 0–5 µM; U2OS 0–40 µM) or etoposide (791T 0–50 µM; HOS 0–50 µM; U2OS 0–1000 µM). Following a one hour drug exposure cells were incubated with or without cobalt chloride for a further 72 hours before fixing and performing a sulphorhodamine-B assay. Graphs show the mean absorbance relative to the untreated controls (UnT) against the log of the drug concentrations and are the average of 3 independent experiments ± SEM. A, Whole cell lysates of cells treated with the above doses of cobalt chloride for the length of the experiment (96 hours) were harvested for western blotting to determine HIF-1α stabilisation and expression of downstream target CA IX. The western blots are representative of 3 independent experiments with GAPDH as a loading control.

Techniques Used: Incubation, Western Blot, Expressing

Hypoxia reduces DNA damage-induced p53 phosphorylation at serine 15, irrespective of HIF-1 inactivation. A, 24 hours after exposure to 1 µM etoposide, 6 µM cisplatin or 0.14 µM doxorubicin for 1 hour in normoxia (N) or hypoxia (H) cells were harvested and western blotting performed for p53, p53 phosphorylated at serine 15, indicative of DNA damage, and the downstream targets of p53, p21, PUMA and NOXA. Treated samples were compared to untreated controls (UnT). GAPDH was a loading control. Data are representative of 3 independent experiments. A reduction of p53 phosphorylation at serine 15 was seen in hypoxia compared to normoxia following exposure to the DNA damaging agents. B, U2OS cells were transiently transfected with the pEF-IRES-P-HIF-no-TAD-EGFP vector (Dominant-negative HIF) (DN) or the empty vector control (EV). After a 24 hour pre-treatment incubation period in either normoxia (N) or hypoxia (H) cells were exposed to 1 µM etoposide and incubated for a further 24 hours before whole cell extracts harvested and western blotting performed for p53, p53 phosphorylated at serine 15, indicative of DNA damage, and p53 transcriptional target p21. Etoposide treated samples were compared to untreated controls. Actin was a loading control. Data are representative of 2 independent experiments. C, Simultaneously transfected cells were maintained in normoxia or hypoxia and harvested at 24 hours hypoxia, the time of treatment. RNA was extracted and qPCR performed for CA IX and Glut-1 expression. Graphs show 2 (−ΔΔCT) where CT is the Cross Threshold and represent the change in mRNA expression in hypoxia relative to normoxia, where 1 would be equivalent expression in normoxia and hypoxia and greater than 1 represents an increase in hypoxia relative to normoxia. Data show mRNA expression from cells lysed in (B) and are representative of 2 independent experiments. Reduced phosphorylation of p53 at serine 15 and p21 protein levels were seen in hypoxia following etoposide treatment despite the inhibition of HIF-1 transcriptional activity.
Figure Legend Snippet: Hypoxia reduces DNA damage-induced p53 phosphorylation at serine 15, irrespective of HIF-1 inactivation. A, 24 hours after exposure to 1 µM etoposide, 6 µM cisplatin or 0.14 µM doxorubicin for 1 hour in normoxia (N) or hypoxia (H) cells were harvested and western blotting performed for p53, p53 phosphorylated at serine 15, indicative of DNA damage, and the downstream targets of p53, p21, PUMA and NOXA. Treated samples were compared to untreated controls (UnT). GAPDH was a loading control. Data are representative of 3 independent experiments. A reduction of p53 phosphorylation at serine 15 was seen in hypoxia compared to normoxia following exposure to the DNA damaging agents. B, U2OS cells were transiently transfected with the pEF-IRES-P-HIF-no-TAD-EGFP vector (Dominant-negative HIF) (DN) or the empty vector control (EV). After a 24 hour pre-treatment incubation period in either normoxia (N) or hypoxia (H) cells were exposed to 1 µM etoposide and incubated for a further 24 hours before whole cell extracts harvested and western blotting performed for p53, p53 phosphorylated at serine 15, indicative of DNA damage, and p53 transcriptional target p21. Etoposide treated samples were compared to untreated controls. Actin was a loading control. Data are representative of 2 independent experiments. C, Simultaneously transfected cells were maintained in normoxia or hypoxia and harvested at 24 hours hypoxia, the time of treatment. RNA was extracted and qPCR performed for CA IX and Glut-1 expression. Graphs show 2 (−ΔΔCT) where CT is the Cross Threshold and represent the change in mRNA expression in hypoxia relative to normoxia, where 1 would be equivalent expression in normoxia and hypoxia and greater than 1 represents an increase in hypoxia relative to normoxia. Data show mRNA expression from cells lysed in (B) and are representative of 2 independent experiments. Reduced phosphorylation of p53 at serine 15 and p21 protein levels were seen in hypoxia following etoposide treatment despite the inhibition of HIF-1 transcriptional activity.

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Dominant Negative Mutation, Incubation, Real-time Polymerase Chain Reaction, Expressing, Inhibition, Activity Assay

Osteosarcoma cells treated with the small molecule inhibitor of HIF-1α NSC134754 remain resistant in hypoxia. A, U2OS cells were treated with 20 µM NSC-134754 for 24 hours in hypoxia prior to exposure to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–4000 µM) for 1 hour. Untreated controls were exposed to the same concentration ranges of cisplatin, doxorubicin and etoposide in normoxia and hypoxia. 72 hours after treatment cells were fixed and a SRB assay performed Graphs show the mean absorbance relative to the untreated controls (no chemotherapy agent) and are the average of 3 independent experiments ± SEM. B, Simultaneously plated cells treated with NSC134754 and incubated in hypoxia for 24 hours (time of treatment) or 96 hours (end of experiment) were harvested for whole cell lysates and western blotting performed for HIF-1α and CA IX. Western blots are representative 3 independent experiments with GAPDH used as a loading control. The difference between the response to cytotoxics in normoxia and hypoxia remains highly significant despite treatment with NSC134754 (p
Figure Legend Snippet: Osteosarcoma cells treated with the small molecule inhibitor of HIF-1α NSC134754 remain resistant in hypoxia. A, U2OS cells were treated with 20 µM NSC-134754 for 24 hours in hypoxia prior to exposure to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–4000 µM) for 1 hour. Untreated controls were exposed to the same concentration ranges of cisplatin, doxorubicin and etoposide in normoxia and hypoxia. 72 hours after treatment cells were fixed and a SRB assay performed Graphs show the mean absorbance relative to the untreated controls (no chemotherapy agent) and are the average of 3 independent experiments ± SEM. B, Simultaneously plated cells treated with NSC134754 and incubated in hypoxia for 24 hours (time of treatment) or 96 hours (end of experiment) were harvested for whole cell lysates and western blotting performed for HIF-1α and CA IX. Western blots are representative 3 independent experiments with GAPDH used as a loading control. The difference between the response to cytotoxics in normoxia and hypoxia remains highly significant despite treatment with NSC134754 (p

Techniques Used: Concentration Assay, Sulforhodamine B Assay, Incubation, Western Blot

Akt is activated in hypoxia in osteosarcoma cells however hypoxia-induced resistance remains despite PI3K inhibition. A, 791T, HOS and U2OS osteosarcoma cells were incubated in normoxia (N) or hypoxia (H) for 48 hours before lysates were analysed for PTEN, Akt and Akt phosphorylated at serine 473 by western blotting. Phosphorylation and therefore activation of Akt is seen in hypoxia in 791T and U2OS cells, coinciding with a reduction of PTEN. B, to determine the effect of PI3K inhibition on drug sensitivity U2OS cells were exposure to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–3000 µM) immediately following treatment with or without 1 µM PI-103 in normoxia and hypoxia. After 72 hours in an SRB assay was performed. The concentration of 1 µM PI-103 was maintained throughout. C, A western blot analysis for Akt and phosphorylated Akt performed after 24 hours hypoxia, at the time of treatment with doxorubicin, and 96 hours hypoxia, at the end of the experiment, showed inhibition of Akt phosphorylation. Despite inhibition of Akt activation, there remained a significant difference in response to cisplatin, doxorubicin and etoposide between normoxia and hypoxia (p
Figure Legend Snippet: Akt is activated in hypoxia in osteosarcoma cells however hypoxia-induced resistance remains despite PI3K inhibition. A, 791T, HOS and U2OS osteosarcoma cells were incubated in normoxia (N) or hypoxia (H) for 48 hours before lysates were analysed for PTEN, Akt and Akt phosphorylated at serine 473 by western blotting. Phosphorylation and therefore activation of Akt is seen in hypoxia in 791T and U2OS cells, coinciding with a reduction of PTEN. B, to determine the effect of PI3K inhibition on drug sensitivity U2OS cells were exposure to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–3000 µM) immediately following treatment with or without 1 µM PI-103 in normoxia and hypoxia. After 72 hours in an SRB assay was performed. The concentration of 1 µM PI-103 was maintained throughout. C, A western blot analysis for Akt and phosphorylated Akt performed after 24 hours hypoxia, at the time of treatment with doxorubicin, and 96 hours hypoxia, at the end of the experiment, showed inhibition of Akt phosphorylation. Despite inhibition of Akt activation, there remained a significant difference in response to cisplatin, doxorubicin and etoposide between normoxia and hypoxia (p

Techniques Used: Inhibition, Incubation, Western Blot, Activation Assay, Sulforhodamine B Assay, Concentration Assay

Osteosarcoma cells expressing dominant-negative HIF-1α remain resistant to cisplatin, doxorubicin and etoposide in hypoxia. U2OS cells were transiently transfected with the pEF-IRES-P-HIF-no-TAD-EGFP vector (Dominant-negative HIF) (DN) or the empty vector control (EV). Following a 24 hour pre-treatment incubation period in either normoxia (N) or hypoxia (H) cells were exposed to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–4000 µM) for 1 hour. 72 hours after treatment cells were fixed and assessed by SRB assay (B). Simultaneously transfected and plated cells were maintained in normoxia or hypoxia and harvested at 24 hours hypoxia (at time of treatment) or 96 hours hypoxia (at the end of the experiment). RNA was extracted and qPCR performed for CA IX and Glut-1 expression (A). Graphs show 2 (−ΔΔCT) where CT is the Cross Threshold and represents the change in mRNA expression in hypoxia relative to normoxia, where 1 would be equivalent expression in normoxia and hypoxia and greater than 1 represents an increase in hypoxia relative to normoxia. Data are the mean ± SEM of 3 independent experiments. * indicates p
Figure Legend Snippet: Osteosarcoma cells expressing dominant-negative HIF-1α remain resistant to cisplatin, doxorubicin and etoposide in hypoxia. U2OS cells were transiently transfected with the pEF-IRES-P-HIF-no-TAD-EGFP vector (Dominant-negative HIF) (DN) or the empty vector control (EV). Following a 24 hour pre-treatment incubation period in either normoxia (N) or hypoxia (H) cells were exposed to a range of concentrations of cisplatin (0–300 µM), doxorubicin (0–100 µM) or etoposide (0–4000 µM) for 1 hour. 72 hours after treatment cells were fixed and assessed by SRB assay (B). Simultaneously transfected and plated cells were maintained in normoxia or hypoxia and harvested at 24 hours hypoxia (at time of treatment) or 96 hours hypoxia (at the end of the experiment). RNA was extracted and qPCR performed for CA IX and Glut-1 expression (A). Graphs show 2 (−ΔΔCT) where CT is the Cross Threshold and represents the change in mRNA expression in hypoxia relative to normoxia, where 1 would be equivalent expression in normoxia and hypoxia and greater than 1 represents an increase in hypoxia relative to normoxia. Data are the mean ± SEM of 3 independent experiments. * indicates p

Techniques Used: Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Incubation, Sulforhodamine B Assay, Real-time Polymerase Chain Reaction

Significant hypoxia-induced resistance remains despite inhibition of HIF-1 by shRNAi or siRNA. A, Stable HOS clones expressing shRNAi to HIF-1α (C5) and firefly luciferase as a control (L4) were incubated in normoxia or hypoxia for 24 hours before exposure to cisplatin (0–150 µM), doxorubicin (0–2.5 µM) or etoposide (0–50 µM) for 1 hour. An SRB assay was performed 72 hours after treatment. B, Western blotting performed on cell lysates from cells simultaneously maintained in hypoxia shows reduced expression of HIF-1α and CA IX, indicating suppressed transcriptional activity, throughout the experiment. D, Stable 791T clones expressing shRNAi to HIF-1α (C24) and firefly luciferase as a control (L3) were similarly processed and treated with doxorubicin (0–48 µM) or etoposide (0–180 µM) for 1 hour. C, Whole cell lysates from cells simultaneously plated were harvested at 24 hours (24H) (at treatment) and 96 hours (96 H) of hypoxia (the experiment end). Western blotting for HIF-1α and CA IX shows suppression of HIF-1α expression and transcriptional activity. E, 791T cells were transiently transfected with siRNA to HIF-1α or a non-targeting control (NT). 8 hours after transfection the hypoxic arm was transferred to hypoxia and after 16 hours cells were treated with cisplatin for 1 hour (0–150 µM). 72 hours after treatment cells were assessed by SRB assay. F, Western blotting on cell lysates collected from cells simultaneously transfected after 24 hours (24 H) and 96 hours (96 H) of hypoxia shows suppression of HIF-1α and CA IX expression. All graphs show the mean absorbance relative to the untreated controls against log drug concentration and are the mean of 3 independent experiments ± SEM. The difference in the drug response of the shRNAi clones and the siRNA transfected cells between hypoxia and normoxia remains highly significant in all cases despite HIF-1α suppression (p
Figure Legend Snippet: Significant hypoxia-induced resistance remains despite inhibition of HIF-1 by shRNAi or siRNA. A, Stable HOS clones expressing shRNAi to HIF-1α (C5) and firefly luciferase as a control (L4) were incubated in normoxia or hypoxia for 24 hours before exposure to cisplatin (0–150 µM), doxorubicin (0–2.5 µM) or etoposide (0–50 µM) for 1 hour. An SRB assay was performed 72 hours after treatment. B, Western blotting performed on cell lysates from cells simultaneously maintained in hypoxia shows reduced expression of HIF-1α and CA IX, indicating suppressed transcriptional activity, throughout the experiment. D, Stable 791T clones expressing shRNAi to HIF-1α (C24) and firefly luciferase as a control (L3) were similarly processed and treated with doxorubicin (0–48 µM) or etoposide (0–180 µM) for 1 hour. C, Whole cell lysates from cells simultaneously plated were harvested at 24 hours (24H) (at treatment) and 96 hours (96 H) of hypoxia (the experiment end). Western blotting for HIF-1α and CA IX shows suppression of HIF-1α expression and transcriptional activity. E, 791T cells were transiently transfected with siRNA to HIF-1α or a non-targeting control (NT). 8 hours after transfection the hypoxic arm was transferred to hypoxia and after 16 hours cells were treated with cisplatin for 1 hour (0–150 µM). 72 hours after treatment cells were assessed by SRB assay. F, Western blotting on cell lysates collected from cells simultaneously transfected after 24 hours (24 H) and 96 hours (96 H) of hypoxia shows suppression of HIF-1α and CA IX expression. All graphs show the mean absorbance relative to the untreated controls against log drug concentration and are the mean of 3 independent experiments ± SEM. The difference in the drug response of the shRNAi clones and the siRNA transfected cells between hypoxia and normoxia remains highly significant in all cases despite HIF-1α suppression (p

Techniques Used: Inhibition, Clone Assay, Expressing, Luciferase, Incubation, Sulforhodamine B Assay, Western Blot, Activity Assay, Transfection, Concentration Assay

Hypoxia leads to cytotoxic drug resistance and reduces cytotoxic-induced apoptosis in osteosarcoma cells. A, Following a 24 hour pre-treatment incubation period in normoxia or hypoxia 791T, HOS and U2OS cells were treated with a range of concentrations of cisplatin (791T 0–150 µM; HOS 0–450 µM; U2OS 0–300 µM), etoposide (791T 0–180 µM; HOS 0–50 µM; U2OS 0–4000 µM) or doxorubicin (791T 0–48 µM; HOS 0–5 µM; U2OS 0–100 µM) for 1 hour. After a further 72 hours an SRB assay was performed. Graphs show the mean absorbance relative to the untreated controls (UnT) against log cisplatin, etoposide or doxorubicin concentration and are the mean ± SEM of 3 independent experiments. The difference between drug response in hypoxia and normoxia is highly significant p
Figure Legend Snippet: Hypoxia leads to cytotoxic drug resistance and reduces cytotoxic-induced apoptosis in osteosarcoma cells. A, Following a 24 hour pre-treatment incubation period in normoxia or hypoxia 791T, HOS and U2OS cells were treated with a range of concentrations of cisplatin (791T 0–150 µM; HOS 0–450 µM; U2OS 0–300 µM), etoposide (791T 0–180 µM; HOS 0–50 µM; U2OS 0–4000 µM) or doxorubicin (791T 0–48 µM; HOS 0–5 µM; U2OS 0–100 µM) for 1 hour. After a further 72 hours an SRB assay was performed. Graphs show the mean absorbance relative to the untreated controls (UnT) against log cisplatin, etoposide or doxorubicin concentration and are the mean ± SEM of 3 independent experiments. The difference between drug response in hypoxia and normoxia is highly significant p

Techniques Used: Incubation, Sulforhodamine B Assay, Concentration Assay

27) Product Images from "Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells"

Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

Journal: Molecular Pharmacology

doi: 10.1124/mol.109.054999

Etoposide triggers caspase-3-dependent apoptosis in SK-N-AS cells. A, cells were treated with 50 μM etoposide for 48 h and harvested. The cytosolic fractions were obtained by a digitonin-based subcellular fractionation procedure. One hundred micrograms of cytosolic (Cy) and mitochondrial protein fractions (Mi) was analyzed by SDS-polyacrylamide gel electrophoresis, and cytochrome c and β-actin levels were determined by immunoblotting. B, immunoblot analysis of caspase-9 and β-actin in cell lysates treated with 50 μM etoposide for 48 h. C, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-9 inhibitor (z-LEHD-fmk) for 48 h. D, immunoblot analysis of PKCδ and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. E, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. F, cells were treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide triggers caspase-3-dependent apoptosis in SK-N-AS cells. A, cells were treated with 50 μM etoposide for 48 h and harvested. The cytosolic fractions were obtained by a digitonin-based subcellular fractionation procedure. One hundred micrograms of cytosolic (Cy) and mitochondrial protein fractions (Mi) was analyzed by SDS-polyacrylamide gel electrophoresis, and cytochrome c and β-actin levels were determined by immunoblotting. B, immunoblot analysis of caspase-9 and β-actin in cell lysates treated with 50 μM etoposide for 48 h. C, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-9 inhibitor (z-LEHD-fmk) for 48 h. D, immunoblot analysis of PKCδ and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. E, immunoblot analysis of caspase-3 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. F, cells were treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Fractionation, Polyacrylamide Gel Electrophoresis, Staining

Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.
Figure Legend Snippet: Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.

Techniques Used: Activation Assay, Expressing

Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Transfection, Staining

Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Activation Assay, Transfection, Staining

Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.
Figure Legend Snippet: Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.

Techniques Used: Transfection, Staining

Etoposide induces PKCδ-mediated apoptosis in SK-N-AS cells. A, immunoblot analysis of PKCδ and β-actin in cell lysates after the treatment with 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were treated as described above, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements. C, immunoblot analysis of PKCδ and β-actin from whole-cell lysates after the transfection with 100 nM nontargeting siRNA (SC) or PKCδ -specific siRNA for 72 h (inset), cells were treated with 50 μM etoposide for an additional 48 h, and apoptosis was determined by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were treated with 10 nM Gö6976 for 2 h followed by 50 μM etoposide for 48 h, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements.
Figure Legend Snippet: Etoposide induces PKCδ-mediated apoptosis in SK-N-AS cells. A, immunoblot analysis of PKCδ and β-actin in cell lysates after the treatment with 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were treated as described above, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements. C, immunoblot analysis of PKCδ and β-actin from whole-cell lysates after the transfection with 100 nM nontargeting siRNA (SC) or PKCδ -specific siRNA for 72 h (inset), cells were treated with 50 μM etoposide for an additional 48 h, and apoptosis was determined by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were treated with 10 nM Gö6976 for 2 h followed by 50 μM etoposide for 48 h, and the percentage of apoptotic cells was determined by staining with annexin V and propidium iodide and analyzed by flow cytometry. All values are representative of three independent experiments, and error bars show S.D. from triplicate measurements.

Techniques Used: Staining, Flow Cytometry, Cytometry, Transfection

Etoposide inhibits the proliferation and triggers apoptosis in SK-N-AS cells. A, cells were treated with 10, 25, 50, or 100 μM etoposide for 48 h, and cell proliferation was determined by using the CellTiter 96 Aqueous One solution cell proliferation assay reagent. Values are representative of three independent experiments, and error bars show S.D. from triplicate counts. B, cells were treated with 50 μM etoposide for 48 h, harvested, and apoptosis was determined by FACScan analysis as described under Materials and Methods . Compensation was executed for each experiment using untreated cells stained with Annexin V and propidium iodide. Error bars show S.D. from triplicate measurements. C, fluorescence microscopy images at 40× magnification of Hoechst 33342-stained nuclei of untreated cells or cells treated with 50 μM etoposide for 48 h. D, quantification of apoptosis was carried out by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.
Figure Legend Snippet: Etoposide inhibits the proliferation and triggers apoptosis in SK-N-AS cells. A, cells were treated with 10, 25, 50, or 100 μM etoposide for 48 h, and cell proliferation was determined by using the CellTiter 96 Aqueous One solution cell proliferation assay reagent. Values are representative of three independent experiments, and error bars show S.D. from triplicate counts. B, cells were treated with 50 μM etoposide for 48 h, harvested, and apoptosis was determined by FACScan analysis as described under Materials and Methods . Compensation was executed for each experiment using untreated cells stained with Annexin V and propidium iodide. Error bars show S.D. from triplicate measurements. C, fluorescence microscopy images at 40× magnification of Hoechst 33342-stained nuclei of untreated cells or cells treated with 50 μM etoposide for 48 h. D, quantification of apoptosis was carried out by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

Techniques Used: Proliferation Assay, Staining, Fluorescence, Microscopy

28) Product Images from "Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway"

Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2018.031

Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p
Figure Legend Snippet: Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

Techniques Used: Cell Culture, Western Blot

Blockade interlukin (IL)-11/IL-11R signal relieves chemotherapy drug resistance in gastric cancer. (A) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in phosphate buffered saline (PBS), doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). Eto, etoposide; Dox, doxorubicin. (B) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (C) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (D) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). (E) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (F) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (G) The schematic diagram of drug resistance development induced by IL-11 in gastric cancer cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p
Figure Legend Snippet: Blockade interlukin (IL)-11/IL-11R signal relieves chemotherapy drug resistance in gastric cancer. (A) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in phosphate buffered saline (PBS), doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). Eto, etoposide; Dox, doxorubicin. (B) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (C) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (D) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). (E) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (F) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (G) The schematic diagram of drug resistance development induced by IL-11 in gastric cancer cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

Techniques Used: Mouse Assay, Injection

Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p
Figure Legend Snippet: Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p

Techniques Used: Expressing, Cell Culture

Cancer-associated-fibroblasts (CAFs) regulated chemo-resistance through secreting interleukin 11 (IL-11). The effect of CAFs on the sensitivity of gastric cancer cells to chemotherapy drugs was examined by using MTT assay. (A) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs medium (CM) pretreated. Dox, doxorubicin; Eto, etoposide. (B) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin. respectively with or without CM pretreated. (C) The mRNA expression of IL-11, SDF-1, HGF, FGF, PDGF, VEGF, and IL-1F9 in normal fibroblast and CAFs. (D) The expression of IL-11R in normal fibroblast and CAFs was detected by using enzyme-linked immunosorbent assay. (E) The mRNA expression of IL-11R, VEGFR, PDGFR, HGFR, CXCR4, SDF-1R, and IL-1F9R in SGC7901 cells with or without CAFs co-cultured. (F) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (G) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 200 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (H) The tumor volume of NOD-SCID mice bearing SGC7901 cells co-injected with CAFs-CM (50 μL 10× CM) or IL-11 (2.5 μg/kg) treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (I) The survival curve of NOD-SCID mice bearing SGC7901 cells co-injected with CAFsCM (50 μL 10× CM) or IL-11 treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (J) Expression of IL-11 in gastric cancer tissues from chemo-sensitive and chemo-resistant patients. The data was presented as the mean±standard error of mean from three independent experiments. * p
Figure Legend Snippet: Cancer-associated-fibroblasts (CAFs) regulated chemo-resistance through secreting interleukin 11 (IL-11). The effect of CAFs on the sensitivity of gastric cancer cells to chemotherapy drugs was examined by using MTT assay. (A) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs medium (CM) pretreated. Dox, doxorubicin; Eto, etoposide. (B) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin. respectively with or without CM pretreated. (C) The mRNA expression of IL-11, SDF-1, HGF, FGF, PDGF, VEGF, and IL-1F9 in normal fibroblast and CAFs. (D) The expression of IL-11R in normal fibroblast and CAFs was detected by using enzyme-linked immunosorbent assay. (E) The mRNA expression of IL-11R, VEGFR, PDGFR, HGFR, CXCR4, SDF-1R, and IL-1F9R in SGC7901 cells with or without CAFs co-cultured. (F) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (G) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 200 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (H) The tumor volume of NOD-SCID mice bearing SGC7901 cells co-injected with CAFs-CM (50 μL 10× CM) or IL-11 (2.5 μg/kg) treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (I) The survival curve of NOD-SCID mice bearing SGC7901 cells co-injected with CAFsCM (50 μL 10× CM) or IL-11 treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (J) Expression of IL-11 in gastric cancer tissues from chemo-sensitive and chemo-resistant patients. The data was presented as the mean±standard error of mean from three independent experiments. * p

Techniques Used: MTT Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Injection

Enriched cancer-associated-fibroblasts (CAFs) enhanced the resistance of gastric cancer cells to chemotherapy. Accumulated CAFs in the chemotherapy-resistant gastric tumor sites and facilitate the resistance to chemotherapy drugs in gastric cancer cells. (A) The percentage of the cancer-associated fibroblasts in samples from chemo-sensitive (CS) and chemo-resistance (CR) gastric cancer patients was detected by flow cytometry. (B) The expression of the cancer-associated fibroblasts in samples from CS and CR gastric cancer patients was detected by immunohistochemistry. (C-H) The cell viability of SGC7901was detected after treated by different concentration of DDP (C), doxorubicin (D), and etoposide (E) pre-co-cultured with or without CAFs by using MTT assay. The cell viability of BGC823 was detected after treated by different concentration of DDP (F), doxorubicin (G), and etoposide (H) pre-co-cultured with or without CAFs by using MTT assay. The data was presented as mean±standard error of mean from three independent experiments. *** p
Figure Legend Snippet: Enriched cancer-associated-fibroblasts (CAFs) enhanced the resistance of gastric cancer cells to chemotherapy. Accumulated CAFs in the chemotherapy-resistant gastric tumor sites and facilitate the resistance to chemotherapy drugs in gastric cancer cells. (A) The percentage of the cancer-associated fibroblasts in samples from chemo-sensitive (CS) and chemo-resistance (CR) gastric cancer patients was detected by flow cytometry. (B) The expression of the cancer-associated fibroblasts in samples from CS and CR gastric cancer patients was detected by immunohistochemistry. (C-H) The cell viability of SGC7901was detected after treated by different concentration of DDP (C), doxorubicin (D), and etoposide (E) pre-co-cultured with or without CAFs by using MTT assay. The cell viability of BGC823 was detected after treated by different concentration of DDP (F), doxorubicin (G), and etoposide (H) pre-co-cultured with or without CAFs by using MTT assay. The data was presented as mean±standard error of mean from three independent experiments. *** p

Techniques Used: Flow Cytometry, Cytometry, Expressing, Immunohistochemistry, Concentration Assay, Cell Culture, MTT Assay

29) Product Images from "CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals"

Article Title: CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals

Journal: Genes & Development

doi: 10.1101/gad.1047003

Serine protease AEBSF inhibits apoptosis induced by etoposide treatment in HeLa cells. HeLa-CIAP1 cells were treated as indicated (un, untreated; ETOP, 10 μM etoposide treatment for 24 h; AE, 100 μg/mL AEBSF treatment for 25 h; AE + ETOP, 100 μg/mL AEBSF was added 1 h prior to additional 10 μM etoposide for another 24 h), followed by TUNEL analysis to determine the apoptotic profile. The results are representative data from two independent experiments.
Figure Legend Snippet: Serine protease AEBSF inhibits apoptosis induced by etoposide treatment in HeLa cells. HeLa-CIAP1 cells were treated as indicated (un, untreated; ETOP, 10 μM etoposide treatment for 24 h; AE, 100 μg/mL AEBSF treatment for 25 h; AE + ETOP, 100 μg/mL AEBSF was added 1 h prior to additional 10 μM etoposide for another 24 h), followed by TUNEL analysis to determine the apoptotic profile. The results are representative data from two independent experiments.

Techniques Used: TUNEL Assay

mRNA of HTRA2 in cultured cells increases upon etoposide treatment or p53 expression. ( A ) Northern blot analysis of HTRA2 mRNA induction upon etoposide treatment. Total RNA was harvested from HeLa cells without or with etoposide treatment for the indicated amount of time. mRNA of HTRA2 or GAPDH genes was detected using 32 P-labeled cDNA probes, quantified by phosphorimager. The fold of induction was normalized against the GAPDH level. ( B ) RT–PCR analysis of HTRA2 and β-actin mRNA from HeLa cells or H1299 cells transfected with p53 expression vector or their respective control (mock-transfected) cells. Intensity of the radioactive species was quantified by PhosphorImager. The fold of induction was normalized against β-actin. These data are representative results from two independent experiments.
Figure Legend Snippet: mRNA of HTRA2 in cultured cells increases upon etoposide treatment or p53 expression. ( A ) Northern blot analysis of HTRA2 mRNA induction upon etoposide treatment. Total RNA was harvested from HeLa cells without or with etoposide treatment for the indicated amount of time. mRNA of HTRA2 or GAPDH genes was detected using 32 P-labeled cDNA probes, quantified by phosphorimager. The fold of induction was normalized against the GAPDH level. ( B ) RT–PCR analysis of HTRA2 and β-actin mRNA from HeLa cells or H1299 cells transfected with p53 expression vector or their respective control (mock-transfected) cells. Intensity of the radioactive species was quantified by PhosphorImager. The fold of induction was normalized against β-actin. These data are representative results from two independent experiments.

Techniques Used: Cell Culture, Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

CIAP1 is cleaved in HeLa cells upon etoposide treatment. ( A ) Silver stain analysis of proteins immunoprecipitated from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ) or etoposide-treated HeLa-CIAP1 cells (lane 3 ). The proteins were identified by MALDI-quadrupole ion trap mass spectrometric analysis using both MS and MS/MS modes. ( B ) MALDI-quadruple mass spectrometric analysis of the two small proteins purified from etoposide-treated HeLa-CIAP1 cell lysate (as indicated in A ). Underlined sequences are tryptic peptides identified from each protein, all mapping to the N-terminal CIAP1 sequence. ( C ) Immunoprecipitated proteins from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ), or etoposide-treated HeLa-CIAP1 cells (lane 3 ), analyzed by immunoblotting with anti-HA antibody. ( D ) Domain structure of CIAP1 and the approximate cleavage sites, as indicated by arrows. BIR, birculoviral IAP Repeat domain; CARD, caspase recruitment domain; Ring Finger domain.
Figure Legend Snippet: CIAP1 is cleaved in HeLa cells upon etoposide treatment. ( A ) Silver stain analysis of proteins immunoprecipitated from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ) or etoposide-treated HeLa-CIAP1 cells (lane 3 ). The proteins were identified by MALDI-quadrupole ion trap mass spectrometric analysis using both MS and MS/MS modes. ( B ) MALDI-quadruple mass spectrometric analysis of the two small proteins purified from etoposide-treated HeLa-CIAP1 cell lysate (as indicated in A ). Underlined sequences are tryptic peptides identified from each protein, all mapping to the N-terminal CIAP1 sequence. ( C ) Immunoprecipitated proteins from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ), or etoposide-treated HeLa-CIAP1 cells (lane 3 ), analyzed by immunoblotting with anti-HA antibody. ( D ) Domain structure of CIAP1 and the approximate cleavage sites, as indicated by arrows. BIR, birculoviral IAP Repeat domain; CARD, caspase recruitment domain; Ring Finger domain.

Techniques Used: Silver Staining, Immunoprecipitation, Mass Spectrometry, Purification, Sequencing

Western blot analysis showing CIAP1 cleavage is associated with p53 induction and is caspase- and apoptosis-independent. Cell lysates from HeLa-CIAP1 with different treatments (as indicated on top: Fas-Ab, treatment of 1 μg/mL Fas antibody for 24 h; Etop, treatment of 10 μM etoposide for 24 h; z-VAD, treatment of 100 μg/mL z-VAD for 25 h; z-VAD + ETOP, treatment of 100 μg/mL z-VAD for 1 h prior to additional 10 μM etoposide treatment another 24 h) were analyzed by immunoblot assay with different antibodies (as indicated on the left side). The identities of each band are indicated to the right . The results are representative data from three independent experiments.
Figure Legend Snippet: Western blot analysis showing CIAP1 cleavage is associated with p53 induction and is caspase- and apoptosis-independent. Cell lysates from HeLa-CIAP1 with different treatments (as indicated on top: Fas-Ab, treatment of 1 μg/mL Fas antibody for 24 h; Etop, treatment of 10 μM etoposide for 24 h; z-VAD, treatment of 100 μg/mL z-VAD for 25 h; z-VAD + ETOP, treatment of 100 μg/mL z-VAD for 1 h prior to additional 10 μM etoposide treatment another 24 h) were analyzed by immunoblot assay with different antibodies (as indicated on the left side). The identities of each band are indicated to the right . The results are representative data from three independent experiments.

Techniques Used: Western Blot

( A ) CIAP1 cleavage requires de novo protein synthesis. ( B ) CIAP1 cleavage induced by etoposide is inhibited by the serine protease inhibitor AEBSF. Cell lysates from HeLa-CIAP1 cells without or with different treatments (as indicated) were immunoblotted with anti-HA antibody, or reprobed with anti-RAN antibody as indicated. The results are representative data from three independent experiments.
Figure Legend Snippet: ( A ) CIAP1 cleavage requires de novo protein synthesis. ( B ) CIAP1 cleavage induced by etoposide is inhibited by the serine protease inhibitor AEBSF. Cell lysates from HeLa-CIAP1 cells without or with different treatments (as indicated) were immunoblotted with anti-HA antibody, or reprobed with anti-RAN antibody as indicated. The results are representative data from three independent experiments.

Techniques Used: Protease Inhibitor

Serine protease inhibitor AEBSF blocks p53-dependent CIAP1 cleavage and apoptosis in primary mouse thymocytes. ( A ) Immunoblot analysis of cell lysates from primary mouse thymocytes [p53 wild type (WT) or p53−/− as indicated] without or with different treatments (ETOP, 20 μM etoposide treatment for 8 h; AEBSF, 100 μg/mL AEBSF was added to the medium 1 h prior to additional 20 μM etoposide treatment for another 8 h) using CIAP1 antibody. ( B ) Apoptotic cell population in primary mouse thymocytes [p53 wild type (WT) or p53−/−, respectively] upon similar treatment in A , as determined by PI staining and Facs analysis. The results are representative data from two independent experiments.
Figure Legend Snippet: Serine protease inhibitor AEBSF blocks p53-dependent CIAP1 cleavage and apoptosis in primary mouse thymocytes. ( A ) Immunoblot analysis of cell lysates from primary mouse thymocytes [p53 wild type (WT) or p53−/− as indicated] without or with different treatments (ETOP, 20 μM etoposide treatment for 8 h; AEBSF, 100 μg/mL AEBSF was added to the medium 1 h prior to additional 20 μM etoposide treatment for another 8 h) using CIAP1 antibody. ( B ) Apoptotic cell population in primary mouse thymocytes [p53 wild type (WT) or p53−/−, respectively] upon similar treatment in A , as determined by PI staining and Facs analysis. The results are representative data from two independent experiments.

Techniques Used: Protease Inhibitor, Staining, FACS

30) Product Images from "DNA damaging agents and p53 do not cause senescence in quiescent cells, while consecutive re-activation of mTOR is associated with conversion to senescence"

Article Title: DNA damaging agents and p53 do not cause senescence in quiescent cells, while consecutive re-activation of mTOR is associated with conversion to senescence

Journal: Aging (Albany NY)

doi:

Serum stimulation of etoposide-locked RPE cells results in mTOR-dependent senescence. A-B . RPE cells were treated with 0.5 μg/ml etoposide in the absence of serum as shown in Figure 6 . Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immunoblotting as indicated ( A ). 4 days after serum stimulation cells were microphotographed ( B ).
Figure Legend Snippet: Serum stimulation of etoposide-locked RPE cells results in mTOR-dependent senescence. A-B . RPE cells were treated with 0.5 μg/ml etoposide in the absence of serum as shown in Figure 6 . Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immunoblotting as indicated ( A ). 4 days after serum stimulation cells were microphotographed ( B ).

Techniques Used: Incubation

Rapamycin pretreatment prevents loss of proliferative potential during etoposide treatment. A - C . WI-38t cells were plated at 10000 cells/well in 24-well plates, and the next day were either left untreated ( A ) or pretreated with 10 nM Rapamycin ( B ). The next day, cells were treated with either 2.5 μM nutlin-3a or 1 μg/ml etoposide or left untreated. After 4 days, cells were trypsinized and 10% of cells were plated in fresh drug-free medium (blue bars). 6 days later cells were counted (red bars). In C the results for etoposide treatment (Et) with or without rapamycin (R) pretreatment are shown in the same scale. ( D ) Cells were lysed after 24 hr treatment with etoposide (E), rapamycin (R), or both (R+E) and immunoblot was performed.
Figure Legend Snippet: Rapamycin pretreatment prevents loss of proliferative potential during etoposide treatment. A - C . WI-38t cells were plated at 10000 cells/well in 24-well plates, and the next day were either left untreated ( A ) or pretreated with 10 nM Rapamycin ( B ). The next day, cells were treated with either 2.5 μM nutlin-3a or 1 μg/ml etoposide or left untreated. After 4 days, cells were trypsinized and 10% of cells were plated in fresh drug-free medium (blue bars). 6 days later cells were counted (red bars). In C the results for etoposide treatment (Et) with or without rapamycin (R) pretreatment are shown in the same scale. ( D ) Cells were lysed after 24 hr treatment with etoposide (E), rapamycin (R), or both (R+E) and immunoblot was performed.

Techniques Used:

Effects of rapamycin and serum starvation on etoposide-induced senescence in RPE cells. A-C. RPE cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 0.5 μg/ml etoposide (Et) was added, as indicated. B. After 4 days, cells were stained for beta-Gal and micro-photographed (bar - 50 micron) C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars corre-spond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 0.5 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin -R, no serum −0.
Figure Legend Snippet: Effects of rapamycin and serum starvation on etoposide-induced senescence in RPE cells. A-C. RPE cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 0.5 μg/ml etoposide (Et) was added, as indicated. B. After 4 days, cells were stained for beta-Gal and micro-photographed (bar - 50 micron) C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars corre-spond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 0.5 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin -R, no serum −0.

Techniques Used: Staining, Incubation

Effects of rapamycin and serum starvation on senescence caused by a higher concentration of etoposide. A. Immunoblot: WI-38t and RPE cells were treated with 0.5 μg/ml and 10 μg/ml etoposide (Et) or left untreated (-). The next day, cells were lysed and immunoblot was performed. B-C: WI-38t and RPE cells were plated at 25000/well in 12 well plates, the next day cells were either pretreated with 10 nM rapamycin (Rapa), placed in serum free medium (no serum) or left in complete medium with 10% serum (control). The next day, 0.5 μg/ml and 10 μg/ml etoposide (Et) was added: in complete medium (control) or with 10 nM Rapamycin (Rapa) or in serum free medium (no serum). After 5 days, cells were washed and cultured in fresh, drug free medium for 11 days and then trypsinized and counted. (in panel C): Before trypsinization, cells treated with 10 μg/ml etoposide (under three conditions: control, Rapa and no serum) were microphotographed.
Figure Legend Snippet: Effects of rapamycin and serum starvation on senescence caused by a higher concentration of etoposide. A. Immunoblot: WI-38t and RPE cells were treated with 0.5 μg/ml and 10 μg/ml etoposide (Et) or left untreated (-). The next day, cells were lysed and immunoblot was performed. B-C: WI-38t and RPE cells were plated at 25000/well in 12 well plates, the next day cells were either pretreated with 10 nM rapamycin (Rapa), placed in serum free medium (no serum) or left in complete medium with 10% serum (control). The next day, 0.5 μg/ml and 10 μg/ml etoposide (Et) was added: in complete medium (control) or with 10 nM Rapamycin (Rapa) or in serum free medium (no serum). After 5 days, cells were washed and cultured in fresh, drug free medium for 11 days and then trypsinized and counted. (in panel C): Before trypsinization, cells treated with 10 μg/ml etoposide (under three conditions: control, Rapa and no serum) were microphotographed.

Techniques Used: Concentration Assay, Cell Culture

Effects of rapamycin and serum starvation on etoposide-induced senescence in WI-38t cells. A-C. WI-38t cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 1 μg/ml etoposide (Et) was added, as indicated. A. After 4 days, cells were stained for beta-Gal and microphotographed (bar - 50 micron). B. After 4 days, cells were counted: control (C), rapamycin (R), no serum (0). C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars correspond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 1 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin - R, no serum −0.
Figure Legend Snippet: Effects of rapamycin and serum starvation on etoposide-induced senescence in WI-38t cells. A-C. WI-38t cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 1 μg/ml etoposide (Et) was added, as indicated. A. After 4 days, cells were stained for beta-Gal and microphotographed (bar - 50 micron). B. After 4 days, cells were counted: control (C), rapamycin (R), no serum (0). C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars correspond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 1 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin - R, no serum −0.

Techniques Used: Staining, Incubation

Experimental schema: transient induction of p53 in proliferating versus quiescent cells Cells are treated (or left untreated) under different condi-tions [control (10% serum), 0% serum or rapamycin] with etoposide for 4 days. Cells are counted twice: 1) at the time of etoposide removal to measure inhibition of proliferation and 2) 6-11 days after wash to measure proliferative potential (PP). PP should not be confused with proliferation. Thus, rapamycin and 0% serum inhibit proliferation but preserve (increase) proliferative potential in etoposide-treated cells.
Figure Legend Snippet: Experimental schema: transient induction of p53 in proliferating versus quiescent cells Cells are treated (or left untreated) under different condi-tions [control (10% serum), 0% serum or rapamycin] with etoposide for 4 days. Cells are counted twice: 1) at the time of etoposide removal to measure inhibition of proliferation and 2) 6-11 days after wash to measure proliferative potential (PP). PP should not be confused with proliferation. Thus, rapamycin and 0% serum inhibit proliferation but preserve (increase) proliferative potential in etoposide-treated cells.

Techniques Used: Inhibition

Serum stimulation of etoposide-locked WI-38t cells results in mTOR-dependent sensecence. A-B . WI38t cells were treated with 1μg/ml etoposide in the absence of serum as shown in Figure 6 . Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immuno-blotting as indicated ( A ). 4 days after serum stimulation cells were microphotographed ( B ).
Figure Legend Snippet: Serum stimulation of etoposide-locked WI-38t cells results in mTOR-dependent sensecence. A-B . WI38t cells were treated with 1μg/ml etoposide in the absence of serum as shown in Figure 6 . Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immuno-blotting as indicated ( A ). 4 days after serum stimulation cells were microphotographed ( B ).

Techniques Used: Incubation

31) Product Images from "CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals"

Article Title: CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mammals

Journal: Genes & Development

doi: 10.1101/gad.1047003

Serine protease AEBSF inhibits apoptosis induced by etoposide treatment in HeLa cells. HeLa-CIAP1 cells were treated as indicated (un, untreated; ETOP, 10 μM etoposide treatment for 24 h; AE, 100 μg/mL AEBSF treatment for 25 h; AE + ETOP, 100 μg/mL AEBSF was added 1 h prior to additional 10 μM etoposide for another 24 h), followed by TUNEL analysis to determine the apoptotic profile. The results are representative data from two independent experiments.
Figure Legend Snippet: Serine protease AEBSF inhibits apoptosis induced by etoposide treatment in HeLa cells. HeLa-CIAP1 cells were treated as indicated (un, untreated; ETOP, 10 μM etoposide treatment for 24 h; AE, 100 μg/mL AEBSF treatment for 25 h; AE + ETOP, 100 μg/mL AEBSF was added 1 h prior to additional 10 μM etoposide for another 24 h), followed by TUNEL analysis to determine the apoptotic profile. The results are representative data from two independent experiments.

Techniques Used: TUNEL Assay

mRNA of HTRA2 in cultured cells increases upon etoposide treatment or p53 expression. ( A ) Northern blot analysis of HTRA2 mRNA induction upon etoposide treatment. Total RNA was harvested from HeLa cells without or with etoposide treatment for the indicated amount of time. mRNA of HTRA2 or GAPDH genes was detected using 32 P-labeled cDNA probes, quantified by phosphorimager. The fold of induction was normalized against the GAPDH level. ( B ) RT–PCR analysis of HTRA2 and β-actin mRNA from HeLa cells or H1299 cells transfected with p53 expression vector or their respective control (mock-transfected) cells. Intensity of the radioactive species was quantified by PhosphorImager. The fold of induction was normalized against β-actin. These data are representative results from two independent experiments.
Figure Legend Snippet: mRNA of HTRA2 in cultured cells increases upon etoposide treatment or p53 expression. ( A ) Northern blot analysis of HTRA2 mRNA induction upon etoposide treatment. Total RNA was harvested from HeLa cells without or with etoposide treatment for the indicated amount of time. mRNA of HTRA2 or GAPDH genes was detected using 32 P-labeled cDNA probes, quantified by phosphorimager. The fold of induction was normalized against the GAPDH level. ( B ) RT–PCR analysis of HTRA2 and β-actin mRNA from HeLa cells or H1299 cells transfected with p53 expression vector or their respective control (mock-transfected) cells. Intensity of the radioactive species was quantified by PhosphorImager. The fold of induction was normalized against β-actin. These data are representative results from two independent experiments.

Techniques Used: Cell Culture, Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

CIAP1 is cleaved in HeLa cells upon etoposide treatment. ( A ) Silver stain analysis of proteins immunoprecipitated from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ) or etoposide-treated HeLa-CIAP1 cells (lane 3 ). The proteins were identified by MALDI-quadrupole ion trap mass spectrometric analysis using both MS and MS/MS modes. ( B ) MALDI-quadruple mass spectrometric analysis of the two small proteins purified from etoposide-treated HeLa-CIAP1 cell lysate (as indicated in A ). Underlined sequences are tryptic peptides identified from each protein, all mapping to the N-terminal CIAP1 sequence. ( C ) Immunoprecipitated proteins from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ), or etoposide-treated HeLa-CIAP1 cells (lane 3 ), analyzed by immunoblotting with anti-HA antibody. ( D ) Domain structure of CIAP1 and the approximate cleavage sites, as indicated by arrows. BIR, birculoviral IAP Repeat domain; CARD, caspase recruitment domain; Ring Finger domain.
Figure Legend Snippet: CIAP1 is cleaved in HeLa cells upon etoposide treatment. ( A ) Silver stain analysis of proteins immunoprecipitated from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ) or etoposide-treated HeLa-CIAP1 cells (lane 3 ). The proteins were identified by MALDI-quadrupole ion trap mass spectrometric analysis using both MS and MS/MS modes. ( B ) MALDI-quadruple mass spectrometric analysis of the two small proteins purified from etoposide-treated HeLa-CIAP1 cell lysate (as indicated in A ). Underlined sequences are tryptic peptides identified from each protein, all mapping to the N-terminal CIAP1 sequence. ( C ) Immunoprecipitated proteins from cell lysates of control untreated HeLa cells (lane 1 ), untreated HeLa-CIAP1 cells (lane 2 ), or etoposide-treated HeLa-CIAP1 cells (lane 3 ), analyzed by immunoblotting with anti-HA antibody. ( D ) Domain structure of CIAP1 and the approximate cleavage sites, as indicated by arrows. BIR, birculoviral IAP Repeat domain; CARD, caspase recruitment domain; Ring Finger domain.

Techniques Used: Silver Staining, Immunoprecipitation, Mass Spectrometry, Purification, Sequencing

Western blot analysis showing CIAP1 cleavage is associated with p53 induction and is caspase- and apoptosis-independent. Cell lysates from HeLa-CIAP1 with different treatments (as indicated on top: Fas-Ab, treatment of 1 μg/mL Fas antibody for 24 h; Etop, treatment of 10 μM etoposide for 24 h; z-VAD, treatment of 100 μg/mL z-VAD for 25 h; z-VAD + ETOP, treatment of 100 μg/mL z-VAD for 1 h prior to additional 10 μM etoposide treatment another 24 h) were analyzed by immunoblot assay with different antibodies (as indicated on the left side). The identities of each band are indicated to the right . The results are representative data from three independent experiments.
Figure Legend Snippet: Western blot analysis showing CIAP1 cleavage is associated with p53 induction and is caspase- and apoptosis-independent. Cell lysates from HeLa-CIAP1 with different treatments (as indicated on top: Fas-Ab, treatment of 1 μg/mL Fas antibody for 24 h; Etop, treatment of 10 μM etoposide for 24 h; z-VAD, treatment of 100 μg/mL z-VAD for 25 h; z-VAD + ETOP, treatment of 100 μg/mL z-VAD for 1 h prior to additional 10 μM etoposide treatment another 24 h) were analyzed by immunoblot assay with different antibodies (as indicated on the left side). The identities of each band are indicated to the right . The results are representative data from three independent experiments.

Techniques Used: Western Blot

( A ) CIAP1 cleavage requires de novo protein synthesis. ( B ) CIAP1 cleavage induced by etoposide is inhibited by the serine protease inhibitor AEBSF. Cell lysates from HeLa-CIAP1 cells without or with different treatments (as indicated) were immunoblotted with anti-HA antibody, or reprobed with anti-RAN antibody as indicated. The results are representative data from three independent experiments.
Figure Legend Snippet: ( A ) CIAP1 cleavage requires de novo protein synthesis. ( B ) CIAP1 cleavage induced by etoposide is inhibited by the serine protease inhibitor AEBSF. Cell lysates from HeLa-CIAP1 cells without or with different treatments (as indicated) were immunoblotted with anti-HA antibody, or reprobed with anti-RAN antibody as indicated. The results are representative data from three independent experiments.

Techniques Used: Protease Inhibitor

Serine protease inhibitor AEBSF blocks p53-dependent CIAP1 cleavage and apoptosis in primary mouse thymocytes. ( A ) Immunoblot analysis of cell lysates from primary mouse thymocytes [p53 wild type (WT) or p53−/− as indicated] without or with different treatments (ETOP, 20 μM etoposide treatment for 8 h; AEBSF, 100 μg/mL AEBSF was added to the medium 1 h prior to additional 20 μM etoposide treatment for another 8 h) using CIAP1 antibody. ( B ) Apoptotic cell population in primary mouse thymocytes [p53 wild type (WT) or p53−/−, respectively] upon similar treatment in A , as determined by PI staining and Facs analysis. The results are representative data from two independent experiments.
Figure Legend Snippet: Serine protease inhibitor AEBSF blocks p53-dependent CIAP1 cleavage and apoptosis in primary mouse thymocytes. ( A ) Immunoblot analysis of cell lysates from primary mouse thymocytes [p53 wild type (WT) or p53−/− as indicated] without or with different treatments (ETOP, 20 μM etoposide treatment for 8 h; AEBSF, 100 μg/mL AEBSF was added to the medium 1 h prior to additional 20 μM etoposide treatment for another 8 h) using CIAP1 antibody. ( B ) Apoptotic cell population in primary mouse thymocytes [p53 wild type (WT) or p53−/−, respectively] upon similar treatment in A , as determined by PI staining and Facs analysis. The results are representative data from two independent experiments.

Techniques Used: Protease Inhibitor, Staining, FACS

32) Product Images from "Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress"

Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038246

Higher expression of specific genes in cells expressing S547A mutated p65. qRT PCR analysis of IL8 ( A ), A20 ( B ), Sele ( C ), VCAM1 ( D ), CXCL1 ( E ) and CD83 ( F ) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .
Figure Legend Snippet: Higher expression of specific genes in cells expressing S547A mutated p65. qRT PCR analysis of IL8 ( A ), A20 ( B ), Sele ( C ), VCAM1 ( D ), CXCL1 ( E ) and CD83 ( F ) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

Techniques Used: Expressing, Quantitative RT-PCR

Higher IL8 protein level in cells expressing S547A mutated p65. ELISA analysis of IL8 protein level after the indicated times of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .
Figure Legend Snippet: Higher IL8 protein level in cells expressing S547A mutated p65. ELISA analysis of IL8 protein level after the indicated times of etoposide treatment in HEK-293 cells expressing either p65 wt or p65 S547A .

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

No impact of p65 Ser 547 phosphorylation on global NF-κB transcriptional activity. ( A ) HEK-293 cells were transfected with κB Luciferase plasmid and with increasing amount of either wt or S547A mutated p65. Twenty-four hours later the cells were treated for 8 h with etoposide or left untreated. NT, non transfected.
Figure Legend Snippet: No impact of p65 Ser 547 phosphorylation on global NF-κB transcriptional activity. ( A ) HEK-293 cells were transfected with κB Luciferase plasmid and with increasing amount of either wt or S547A mutated p65. Twenty-four hours later the cells were treated for 8 h with etoposide or left untreated. NT, non transfected.

Techniques Used: Activity Assay, Transfection, Luciferase, Plasmid Preparation

Creation of HEK-293 cell lines expressing either wt or S547A mutated p65. ( A ) Schematic representation of the 2 silent mutations inserted in the sequence of p65 within the sequence targeted by p65 siRNA, conferring to exogenous HA-p65 the resistance to the siRNA. ( B ) Western blot analysis of cytoplasmic and nuclear p65 protein (p65 antibody) and of exogenous p65 (HA antibody) in non-treated or etoposide treated HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were either transfected with control or p65 siRNA. ( C–D ) Comparison of NF-κB activation after etoposide treatment, by (C) EMSA and by (D) qRT PCR of IκBα mRNA, between HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were transfected either with control or p65 siRNA. ( C ) NF-κB DNA binding was analyzed using a probe corresponding to the HIV-1 long terminal repeat κB site (top panel). The presence of p65 and p50 protein in the binding complex observed in HEK-293 was analyzed by supershift with p65 and p50 antibodies (bottom panel). ns, non specific band.
Figure Legend Snippet: Creation of HEK-293 cell lines expressing either wt or S547A mutated p65. ( A ) Schematic representation of the 2 silent mutations inserted in the sequence of p65 within the sequence targeted by p65 siRNA, conferring to exogenous HA-p65 the resistance to the siRNA. ( B ) Western blot analysis of cytoplasmic and nuclear p65 protein (p65 antibody) and of exogenous p65 (HA antibody) in non-treated or etoposide treated HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were either transfected with control or p65 siRNA. ( C–D ) Comparison of NF-κB activation after etoposide treatment, by (C) EMSA and by (D) qRT PCR of IκBα mRNA, between HEK-293 and HEK-293 stably expressing sip65 resistant HA-p65. All cell lines were transfected either with control or p65 siRNA. ( C ) NF-κB DNA binding was analyzed using a probe corresponding to the HIV-1 long terminal repeat κB site (top panel). The presence of p65 and p50 protein in the binding complex observed in HEK-293 was analyzed by supershift with p65 and p50 antibodies (bottom panel). ns, non specific band.

Techniques Used: Expressing, Sequencing, Western Blot, Stable Transfection, Transfection, Activation Assay, Quantitative RT-PCR, Binding Assay

Ser 547 phosphorylation–dependent IL8 gene regulation mechanism involve HDAC. ( A–B ) HEK-293 expressing either p65 wt or p65 S547A were non-treated or treated 4 hours with etoposide in absence or presence of TSA, and mRNA levels of IL8 ( A ) and IκBα ( B ) were analyzed by qRT PCR. ( C–D ) Recruitments of Lys 9 acetylated histone 3 on IL8 ( C ) and IκBα ( D ) promoters were measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ( E ) HA-tagged p65 wt or p65 S547A were immunoprecipitated from lysates of HEK-293 cell transfected with expression plasmid for these two proteins or non transfected as control. Co-immunoprecipitated HDAC1 protein was detected by western blotting (upper panel). Level of immunoprecipitated HA-p65 in the different samples was determined by western blotting (lower panel). A portion of whole cell extract was added on the gel as input. ( F ) Recruitment of HDAC1 on IL8 promoter was measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated 5 hours with etoposide or left untreated. *, significantly different (P
Figure Legend Snippet: Ser 547 phosphorylation–dependent IL8 gene regulation mechanism involve HDAC. ( A–B ) HEK-293 expressing either p65 wt or p65 S547A were non-treated or treated 4 hours with etoposide in absence or presence of TSA, and mRNA levels of IL8 ( A ) and IκBα ( B ) were analyzed by qRT PCR. ( C–D ) Recruitments of Lys 9 acetylated histone 3 on IL8 ( C ) and IκBα ( D ) promoters were measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ( E ) HA-tagged p65 wt or p65 S547A were immunoprecipitated from lysates of HEK-293 cell transfected with expression plasmid for these two proteins or non transfected as control. Co-immunoprecipitated HDAC1 protein was detected by western blotting (upper panel). Level of immunoprecipitated HA-p65 in the different samples was determined by western blotting (lower panel). A portion of whole cell extract was added on the gel as input. ( F ) Recruitment of HDAC1 on IL8 promoter was measured by ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated 5 hours with etoposide or left untreated. *, significantly different (P

Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot

Similar etoposide-induced gene expression profile in cells expressing wt and S547D mutated p65. qRT PCR analysis of IL8 (A), and IκBα (B) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt , p65 S547A , or p65 S547D .
Figure Legend Snippet: Similar etoposide-induced gene expression profile in cells expressing wt and S547D mutated p65. qRT PCR analysis of IL8 (A), and IκBα (B) mRNA level after 4 and 8 hours of etoposide treatment in HEK-293 cells expressing either p65 wt , p65 S547A , or p65 S547D .

Techniques Used: Expressing, Quantitative RT-PCR

Identical binding of wt and S547A mutated p65 to IL8 κB site. ( A ) NF-κB binding to IL8 promoter, in HEK-293 cells expressing either p65 wt or p65 S547A , non-treated or treated with etoposide, was analyzed by EMSA with a probe corresponding to the κB site of IL8 promoter. ( B ) The presence of p65 protein in the binding complex observed in ( A ) was analyzed by supershift with p65 antibody. ( C ) Recruitment of p65 on IL8 promoter was measured by p65 ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ns, non significantly different.
Figure Legend Snippet: Identical binding of wt and S547A mutated p65 to IL8 κB site. ( A ) NF-κB binding to IL8 promoter, in HEK-293 cells expressing either p65 wt or p65 S547A , non-treated or treated with etoposide, was analyzed by EMSA with a probe corresponding to the κB site of IL8 promoter. ( B ) The presence of p65 protein in the binding complex observed in ( A ) was analyzed by supershift with p65 antibody. ( C ) Recruitment of p65 on IL8 promoter was measured by p65 ChIP assay in HEK-293 cells expressing either p65 wt or p65 S547A , and treated with etoposide for the indicated periods. ns, non significantly different.

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation

33) Product Images from "Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage"

Article Title: Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.1.28-38.2006

Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in primary human cells. HMEC were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. (A and C) Cell viability was measured at 48 h; (B and D) cell proliferation at 48 h was measured by monitoring [ 14 C]thymidine incorporation.
Figure Legend Snippet: Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in primary human cells. HMEC were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. (A and C) Cell viability was measured at 48 h; (B and D) cell proliferation at 48 h was measured by monitoring [ 14 C]thymidine incorporation.

Techniques Used: Inhibition, Activity Assay

Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in cell lines. NCI-H460, U-2 OS, and MCF-7 cells were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. NCI-H460 cells were additionally treated with 5 mM nicotinamide. (A, C, and E) Cell viability was measured at 48 h; (B, D, and F) cell proliferation at 72 h was measured by monitoring [ 14 C]thymidine incorporation.
Figure Legend Snippet: Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in cell lines. NCI-H460, U-2 OS, and MCF-7 cells were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. NCI-H460 cells were additionally treated with 5 mM nicotinamide. (A, C, and E) Cell viability was measured at 48 h; (B, D, and F) cell proliferation at 72 h was measured by monitoring [ 14 C]thymidine incorporation.

Techniques Used: Inhibition, Activity Assay

Inhibition of SIRT1 catalytic activity increases p53 acetylation after DNA damage. (A) Effect of EX-527 on p53 acetylation after DNA damage. NCI-H460 cells were treated with etoposide in combination with either nicotinamide (Nicot.), EX-527, or dimethyl sulfoxide vehicle (DMSO) for 6 h. Blots were probed with an anti-acetylated p53 Lys 382 antibody (upper panel) or p53 antibody (Ab-7) that recognizes all forms of human p53 (lower panel). (B) Effect of EX-527 and a series of closely related compounds on p53 acetylation after DNA damage. Cells were treated with 1 μM concentrations of the indicated compounds for 6 h, and samples were prepared as described for panel A, except that cells were treated additionally with 25 nM TSA to inhibit class I/II HDAC activity. (C) Effect of enantiomers of EX-527 on p53 acetylation after DNA damage. Cells were treated with EX-242 or EX-243 for 6 h. p53 was immunoprecipitated and immunoblotted as described for panel A. +, present; −, absent.
Figure Legend Snippet: Inhibition of SIRT1 catalytic activity increases p53 acetylation after DNA damage. (A) Effect of EX-527 on p53 acetylation after DNA damage. NCI-H460 cells were treated with etoposide in combination with either nicotinamide (Nicot.), EX-527, or dimethyl sulfoxide vehicle (DMSO) for 6 h. Blots were probed with an anti-acetylated p53 Lys 382 antibody (upper panel) or p53 antibody (Ab-7) that recognizes all forms of human p53 (lower panel). (B) Effect of EX-527 and a series of closely related compounds on p53 acetylation after DNA damage. Cells were treated with 1 μM concentrations of the indicated compounds for 6 h, and samples were prepared as described for panel A, except that cells were treated additionally with 25 nM TSA to inhibit class I/II HDAC activity. (C) Effect of enantiomers of EX-527 on p53 acetylation after DNA damage. Cells were treated with EX-242 or EX-243 for 6 h. p53 was immunoprecipitated and immunoblotted as described for panel A. +, present; −, absent.

Techniques Used: Inhibition, Activity Assay, Immunoprecipitation

Inhibition of SIRT1 catalytic activity enhances p53 lysine 382 acetylation after a variety of DNA-damaging agents and is not cell type specific. (A) NCI-H460 cells were treated with adriamycin, hydroxyurea, or hydrogen peroxide for 6 h. p53 was immunoprecipitated and immunoblotted with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). (B) U-2 OS and MCF-7 cells were treated with etoposide in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting was performed as described for panel A. (C) HMEC were treated with etoposide or adriamycin in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting of p53 was performed as described for panel A. Total SIRT1 levels were measured by immunoblotting using antibody 07-131 (Upstate Cell Signaling Solutions). +, present; −, absent.
Figure Legend Snippet: Inhibition of SIRT1 catalytic activity enhances p53 lysine 382 acetylation after a variety of DNA-damaging agents and is not cell type specific. (A) NCI-H460 cells were treated with adriamycin, hydroxyurea, or hydrogen peroxide for 6 h. p53 was immunoprecipitated and immunoblotted with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). (B) U-2 OS and MCF-7 cells were treated with etoposide in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting was performed as described for panel A. (C) HMEC were treated with etoposide or adriamycin in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting of p53 was performed as described for panel A. Total SIRT1 levels were measured by immunoblotting using antibody 07-131 (Upstate Cell Signaling Solutions). +, present; −, absent.

Techniques Used: Inhibition, Activity Assay, Immunoprecipitation

p53-controlled gene expression is not altered by inhibition of SIRT1 in several p53-positive cell lines. (A) NCI-H460, MCF-7, U-2 OS, and HMEC were treated with various concentrations of etoposide for 6 h in the presence of 1 μM EX-527 or dimethyl sulfoxide vehicle (DMSO). Levels of p21 mRNA were measured using quantitative PCR and normalized to the expression levels of hypoxanthine phosphoribosyltransferase (HPRT). (B) NCI-H460 cells were treated with etoposide or adriamycin in the presence or absence of EX-527 for 6 h. Cell lysates were prepared for immunoblotting and were probed with antibodies directed against p21 and tubulin. +, present; −, absent.
Figure Legend Snippet: p53-controlled gene expression is not altered by inhibition of SIRT1 in several p53-positive cell lines. (A) NCI-H460, MCF-7, U-2 OS, and HMEC were treated with various concentrations of etoposide for 6 h in the presence of 1 μM EX-527 or dimethyl sulfoxide vehicle (DMSO). Levels of p21 mRNA were measured using quantitative PCR and normalized to the expression levels of hypoxanthine phosphoribosyltransferase (HPRT). (B) NCI-H460 cells were treated with etoposide or adriamycin in the presence or absence of EX-527 for 6 h. Cell lysates were prepared for immunoblotting and were probed with antibodies directed against p21 and tubulin. +, present; −, absent.

Techniques Used: Expressing, Inhibition, Real-time Polymerase Chain Reaction

Effect of inhibiting SIRT1 and HDAC1 on p53 lysine 382 acetylation and cell growth. (A) NCI-H460 cells were treated for 6 h with various concentrations of TSA and 1 μM EX-527 in the presence of 20 μM etoposide. Cell lysates were analyzed by immunoblotting with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). The graph shows the quantification of bands on the gel shown, but this is representative of the results from two such experiments. +, present; −, absent. (B to E) NCI-H460 cells were treated with increasing concentrations of etoposide or adriamycin and deacetylase inhibitors as indicated. Dimethyl sulfoxide (DMSO) was used as the vehicle control. (B and D) Cell proliferation at 72 h was measured by monitoring [ 14 C]thymidine incorporation; (C and E) cell viability was measured at 48 h.
Figure Legend Snippet: Effect of inhibiting SIRT1 and HDAC1 on p53 lysine 382 acetylation and cell growth. (A) NCI-H460 cells were treated for 6 h with various concentrations of TSA and 1 μM EX-527 in the presence of 20 μM etoposide. Cell lysates were analyzed by immunoblotting with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). The graph shows the quantification of bands on the gel shown, but this is representative of the results from two such experiments. +, present; −, absent. (B to E) NCI-H460 cells were treated with increasing concentrations of etoposide or adriamycin and deacetylase inhibitors as indicated. Dimethyl sulfoxide (DMSO) was used as the vehicle control. (B and D) Cell proliferation at 72 h was measured by monitoring [ 14 C]thymidine incorporation; (C and E) cell viability was measured at 48 h.

Techniques Used: Histone Deacetylase Assay

34) Product Images from "Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells"

Article Title: Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

Journal: Molecular Biology of the Cell

doi:

Schematic representation of the structure of Cas with the predicted cleavage sites and the functional domains. The following regions within Cas are indicated: the SH3 domain (SH3), the proline-rich sequence (Pro), the substrate domain (YXXP, tyrosine phosphorylation sites), and the serine-rich domain (SR), which includes multiple serine phosphorylation consensus motifs, the Src binding site (SBS), a proline-rich sequence that associates with the SH3 domain, and a tyrosine residue that upon phosphorylation binds to the SH2 domain. The immunogens for Cas mAb and Cas-2 Ab are illustrated. Ten DXXD sequences conserved in rat and mouse p130 Cas are indicated (209). The putative fragments (AF1 and AF2) generated by caspase-3-catalyzed cleavage during etoposide-induced apoptosis are indicated (τ, putative cleavage site).
Figure Legend Snippet: Schematic representation of the structure of Cas with the predicted cleavage sites and the functional domains. The following regions within Cas are indicated: the SH3 domain (SH3), the proline-rich sequence (Pro), the substrate domain (YXXP, tyrosine phosphorylation sites), and the serine-rich domain (SR), which includes multiple serine phosphorylation consensus motifs, the Src binding site (SBS), a proline-rich sequence that associates with the SH3 domain, and a tyrosine residue that upon phosphorylation binds to the SH2 domain. The immunogens for Cas mAb and Cas-2 Ab are illustrated. Ten DXXD sequences conserved in rat and mouse p130 Cas are indicated (209). The putative fragments (AF1 and AF2) generated by caspase-3-catalyzed cleavage during etoposide-induced apoptosis are indicated (τ, putative cleavage site).

Techniques Used: Functional Assay, Sequencing, Binding Assay, Generated

In vivo cleavage of mutant Cas. Rat-1 cells transiently transfected with pFLAG-CMV-5c containing each mutant Cas DNA such as vector only (lane 1), wild-type Cas (wt, lanes 2 and 4), single-mutant Cas (D 416 E, lane 5; and D 748 E, lane 6), and double-mutant Cas (D 416 E and D 748 E, lanes 3 and 7) were cultured with (lanes 4- 7) or without (lanes 1–3) etoposide for 24 h. The cell lysates were immunoblotted with anti-FLAG M2 mAb (A), and the same membrane was then stripped and reprobed with Cas mAb (B). Note that the overexpresed mutant Cas were resistant to in vivo cleavage. Molecular mass standards are shown by the arrowheads on the left. Molecular masses of the cleavage products are shown by the arrows on the right. Nonspecific binding of antibodies is indicated by closed squares on the right.
Figure Legend Snippet: In vivo cleavage of mutant Cas. Rat-1 cells transiently transfected with pFLAG-CMV-5c containing each mutant Cas DNA such as vector only (lane 1), wild-type Cas (wt, lanes 2 and 4), single-mutant Cas (D 416 E, lane 5; and D 748 E, lane 6), and double-mutant Cas (D 416 E and D 748 E, lanes 3 and 7) were cultured with (lanes 4- 7) or without (lanes 1–3) etoposide for 24 h. The cell lysates were immunoblotted with anti-FLAG M2 mAb (A), and the same membrane was then stripped and reprobed with Cas mAb (B). Note that the overexpresed mutant Cas were resistant to in vivo cleavage. Molecular mass standards are shown by the arrowheads on the left. Molecular masses of the cleavage products are shown by the arrows on the right. Nonspecific binding of antibodies is indicated by closed squares on the right.

Techniques Used: In Vivo, Mutagenesis, Transfection, Plasmid Preparation, Cell Culture, Binding Assay

Fluorescent images depicting the changes in the cellular localization of Cas, paxillin, talin, and actin in apoptotic cells. The distribution of Cas (4F4, A and B), paxillin (C and D), and talin (E and F) within control cells or etoposide-treated cells reveals that Cas, paxillin, and talin are localized in focal adhesion sites in control cells (A, C, and E) but is lost from focal adhesions during apoptosis and redistributes into the periphery of cells (B, D, and F). Actin labeling using TRITC-phalloidin reveals that the actin stress fibers seen in control cells (G) are virtually absent from the cytoplasm of apoptotic cells, although some truncated fibers are seen at the cell margins (H). Bar, 10 μm.
Figure Legend Snippet: Fluorescent images depicting the changes in the cellular localization of Cas, paxillin, talin, and actin in apoptotic cells. The distribution of Cas (4F4, A and B), paxillin (C and D), and talin (E and F) within control cells or etoposide-treated cells reveals that Cas, paxillin, and talin are localized in focal adhesion sites in control cells (A, C, and E) but is lost from focal adhesions during apoptosis and redistributes into the periphery of cells (B, D, and F). Actin labeling using TRITC-phalloidin reveals that the actin stress fibers seen in control cells (G) are virtually absent from the cytoplasm of apoptotic cells, although some truncated fibers are seen at the cell margins (H). Bar, 10 μm.

Techniques Used: Labeling

Inhibition of Cas proteolysis in vivo by ZAVD-fmk and DEVD-cmk. (A) Rat-1 cells were pretreated for 3 h with the indicated concentrations of ZVAD-fmk or DEVD-cmk and then exposed to 40 μM etoposide for an additional 12 h. Cas proteolysis was analyzed by immunoblot analysis using Cas mAb as a probe. The same membrane was then stripped and reprobed with FAK mAb. Both antagonists effectively inhibited Cas and FAK cleavage. Molecular mass standards are indicated by the small arrowheads on the left. Original proteins (arrows) and cleavage fragments (large arrowheads) are shown on the right. (B) Apoptotic cell lysates (30 μg) were obtained from etoposide-treated cells for 36 h and incubated with in vitro–translated Cas in the presence of either ZVAD-fmk or DEVD-cmk. Both caspase inhibitors completely inhibited the cleavage of in vitro–translated Cas even at 50 nM.
Figure Legend Snippet: Inhibition of Cas proteolysis in vivo by ZAVD-fmk and DEVD-cmk. (A) Rat-1 cells were pretreated for 3 h with the indicated concentrations of ZVAD-fmk or DEVD-cmk and then exposed to 40 μM etoposide for an additional 12 h. Cas proteolysis was analyzed by immunoblot analysis using Cas mAb as a probe. The same membrane was then stripped and reprobed with FAK mAb. Both antagonists effectively inhibited Cas and FAK cleavage. Molecular mass standards are indicated by the small arrowheads on the left. Original proteins (arrows) and cleavage fragments (large arrowheads) are shown on the right. (B) Apoptotic cell lysates (30 μg) were obtained from etoposide-treated cells for 36 h and incubated with in vitro–translated Cas in the presence of either ZVAD-fmk or DEVD-cmk. Both caspase inhibitors completely inhibited the cleavage of in vitro–translated Cas even at 50 nM.

Techniques Used: Inhibition, In Vivo, Incubation, In Vitro

Proteolytic cleavage of Cas during etoposide-induced apoptosis in Rat-1 cells. Rat-1 cells were exposed to 40 μM etoposide for the indicated periods. Cell lysates were then subjected to immunoblot analysis using Cas mAb (A) and Cas-2 (B). Intact Cas migrates at a molecular mass of 130-kDa. Cleavage products of Cas are shown (large arrowheads on the right; 31 and 47 kDa). The blot was then reprobed with FAK mAb (C), after which, to verify equal loading of protein, the blot was stripped once again and reprobed with tubulin mAb (D). Molecular mass standards are indicated by the small arrowheads on the left.
Figure Legend Snippet: Proteolytic cleavage of Cas during etoposide-induced apoptosis in Rat-1 cells. Rat-1 cells were exposed to 40 μM etoposide for the indicated periods. Cell lysates were then subjected to immunoblot analysis using Cas mAb (A) and Cas-2 (B). Intact Cas migrates at a molecular mass of 130-kDa. Cleavage products of Cas are shown (large arrowheads on the right; 31 and 47 kDa). The blot was then reprobed with FAK mAb (C), after which, to verify equal loading of protein, the blot was stripped once again and reprobed with tubulin mAb (D). Molecular mass standards are indicated by the small arrowheads on the left.

Techniques Used:

Changes of cellular localization of Cas in double-mutant Cas-transfected Rat-1 cells. Rat-1 cells were transiently transfected with pFLAG-CMV-5c containing double-mutant (D 416 E and D 748 E) or wild-type Cas DNAs and incubated with 40 μM etoposide for 18 h (A– D) or 24 h (E and F). Cells were double immunostained with anti-FLAG M2 mAb (A, C, and E) and paxillin Ab (B, D, and F). Positive staining with anti-FLAG M2 mAb indicates cells expressing double-mutant or wild-type Cas. The images in A, C, and E depict alternate staining of the same cells shown in B, D, and F, respectively. Focal adhesions at the cell bottom are indicated by white arrows. Note that double-mutant Cas could attenuate the Cas degradation and consequently partially blocked the relocalization of focal adhesion proteins into the periphery of cells. Bar, 10 μm.
Figure Legend Snippet: Changes of cellular localization of Cas in double-mutant Cas-transfected Rat-1 cells. Rat-1 cells were transiently transfected with pFLAG-CMV-5c containing double-mutant (D 416 E and D 748 E) or wild-type Cas DNAs and incubated with 40 μM etoposide for 18 h (A– D) or 24 h (E and F). Cells were double immunostained with anti-FLAG M2 mAb (A, C, and E) and paxillin Ab (B, D, and F). Positive staining with anti-FLAG M2 mAb indicates cells expressing double-mutant or wild-type Cas. The images in A, C, and E depict alternate staining of the same cells shown in B, D, and F, respectively. Focal adhesions at the cell bottom are indicated by white arrows. Note that double-mutant Cas could attenuate the Cas degradation and consequently partially blocked the relocalization of focal adhesion proteins into the periphery of cells. Bar, 10 μm.

Techniques Used: Mutagenesis, Transfection, Incubation, Staining, Expressing

35) Product Images from "p53-mediated delayed NF-κB activity enhances etoposide-induced cell death in medulloblastoma"

Article Title: p53-mediated delayed NF-κB activity enhances etoposide-induced cell death in medulloblastoma

Journal: Cell Death & Disease

doi: 10.1038/cddis.2010.16

p65 phosphorylation is induced by a p53-dependent death receptors expression. ( a ) Death receptor expression was measured in all cell lines by qPCR upon 8 h etoposide treatment (20 μ M). The plot represents the relative quantification compared to control non-treated cells. ( b ) Fas receptor immunocytochemistry on D283-MED cells treated with 20 μ M etoposide for 8 h. ( c ) Quantification by flow cytometry of Fas expression to the plasma membrane in D283-MED cells upon 8 h of etoposide treatment (20 μ M). ( d ) D283-MED cells were transfected with 100 nM of siRNA directed against p53 or control-scrambled siRNA. After 48 h transfection, cells were treated with etoposide (20 μ M, 8 h). Fas mRNA expression was assessed by qPCR. Results are expressed as fold levels induction compared with the control unstimulated cells transfected with scrambled siRNA. Knockdown of p53 was controlled by qPCR (not shown) and western blot ( Figure 3c ). ( e ) D283-MED cells were transfected with 100 nM siRNA directed against Fas or control-scrambled siRNA. After 48 h transfection, cells were treated with 20 μ M etoposide for 6 h and phospho-p65 levels were measured by western blot. The plot represents the quantification of the blot shown. ( f ) D283-MED cells were treated with 20 μ M etoposide in the presence or absence of the Fas antagonist ZB4 antibody (5 μ g/ml) for 8 h. Cell viability was measured by MTS assay and expressed as % of control untreated cells. ( g ) D283-MED cells were treated for 6 h with etoposide (20 μ M) in the presence or absence of the Fas antagonist ZB4 antibody. Phospho-Ser536-p65 levels were evaluated by western blot. The bands were quantified and the phospho-p65/p65 ratio was plotted for the different treatment conditions
Figure Legend Snippet: p65 phosphorylation is induced by a p53-dependent death receptors expression. ( a ) Death receptor expression was measured in all cell lines by qPCR upon 8 h etoposide treatment (20 μ M). The plot represents the relative quantification compared to control non-treated cells. ( b ) Fas receptor immunocytochemistry on D283-MED cells treated with 20 μ M etoposide for 8 h. ( c ) Quantification by flow cytometry of Fas expression to the plasma membrane in D283-MED cells upon 8 h of etoposide treatment (20 μ M). ( d ) D283-MED cells were transfected with 100 nM of siRNA directed against p53 or control-scrambled siRNA. After 48 h transfection, cells were treated with etoposide (20 μ M, 8 h). Fas mRNA expression was assessed by qPCR. Results are expressed as fold levels induction compared with the control unstimulated cells transfected with scrambled siRNA. Knockdown of p53 was controlled by qPCR (not shown) and western blot ( Figure 3c ). ( e ) D283-MED cells were transfected with 100 nM siRNA directed against Fas or control-scrambled siRNA. After 48 h transfection, cells were treated with 20 μ M etoposide for 6 h and phospho-p65 levels were measured by western blot. The plot represents the quantification of the blot shown. ( f ) D283-MED cells were treated with 20 μ M etoposide in the presence or absence of the Fas antagonist ZB4 antibody (5 μ g/ml) for 8 h. Cell viability was measured by MTS assay and expressed as % of control untreated cells. ( g ) D283-MED cells were treated for 6 h with etoposide (20 μ M) in the presence or absence of the Fas antagonist ZB4 antibody. Phospho-Ser536-p65 levels were evaluated by western blot. The bands were quantified and the phospho-p65/p65 ratio was plotted for the different treatment conditions

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry, Flow Cytometry, Cytometry, Transfection, Western Blot, MTS Assay

The upstream role of p53 in cell death sensitivity is also valid in glioblastoma cells. ( a ) Cell viability was analysed in several glioblastoma cell lines by MTS assay after 24 h of 20 μ M etoposide treatment. ( b ) Glioblastoma cells were treated with 20 μ M etoposide for 6 h and p53 expression levels as well as p65 phosphorylation levels were evaluated by western blot in different cell lines. The bands were quantified and the fold induction of phospho-Ser536-p65 (p65 as loading control) and p53 expression (actin as loading control) normalised to the control unstimulated cells were plotted for the different treatment conditions. ( c ) Glioblastoma cells were treated with 20 μ M etoposide for 8 h and mRNA level of p53-dependent genes (mdm2 and Fas) and NF- κ B-dependent genes (I κ B α and A20) were assessed by qPCR. Results are expressed as fold-induction compared with the control unstimulated cells. ( d ) Glioblastoma cells transfected with p65-RedXP and I κ B α -EGFP were treated with 10 ng/ml TNF α . Time-lapse confocal microscopy was performed as described in experimental procedures. Mean fluorescence intensities for individual cells were analysed. A typical cell for each cell line was plotted as the p65 nuclear/cytoplasmic ratio as a function of time. Pictures illustrate a typical D566-MG cell at indicated time points. The scale bar represents 20 μ m
Figure Legend Snippet: The upstream role of p53 in cell death sensitivity is also valid in glioblastoma cells. ( a ) Cell viability was analysed in several glioblastoma cell lines by MTS assay after 24 h of 20 μ M etoposide treatment. ( b ) Glioblastoma cells were treated with 20 μ M etoposide for 6 h and p53 expression levels as well as p65 phosphorylation levels were evaluated by western blot in different cell lines. The bands were quantified and the fold induction of phospho-Ser536-p65 (p65 as loading control) and p53 expression (actin as loading control) normalised to the control unstimulated cells were plotted for the different treatment conditions. ( c ) Glioblastoma cells were treated with 20 μ M etoposide for 8 h and mRNA level of p53-dependent genes (mdm2 and Fas) and NF- κ B-dependent genes (I κ B α and A20) were assessed by qPCR. Results are expressed as fold-induction compared with the control unstimulated cells. ( d ) Glioblastoma cells transfected with p65-RedXP and I κ B α -EGFP were treated with 10 ng/ml TNF α . Time-lapse confocal microscopy was performed as described in experimental procedures. Mean fluorescence intensities for individual cells were analysed. A typical cell for each cell line was plotted as the p65 nuclear/cytoplasmic ratio as a function of time. Pictures illustrate a typical D566-MG cell at indicated time points. The scale bar represents 20 μ m

Techniques Used: MTS Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Confocal Microscopy, Fluorescence

MB cell lines displayed different sensitivity to etoposide involving both caspase-dependent and -independent cell death. ( a ) Cell viability was analysed by MTS assay at indicated time points, upon 20 μ M etoposide treatment. ( b ) Viability of D283-MED cells treated with 20 μ M etoposide in the presence of 5 μ M Bay11-7082, 10 μ M Wedelolactone or 5 μ M JSH23 was assessed by MTS assay. ( c ) D283-Med cells were transfected with 100 nM of siRNA directed against p65 or a control scrambled siRNA. After 48 h, cells were treated with 20 μ M etoposide for 8 h and viability was assessed by MTS assay. The knockdown efficiency was controlled by western blot (insert). ( d ) Several MB cell lines were treated with 20 μ M etoposide for indicated times. Caspase 3/7 activity was measured with Caspase-Glo 3/7 assay kit. Results are expressed as fold-induction compared with control non-treated cells as a function of time. ( e ) D283-MED cells were treated with 20 μ M etoposide in presence or absence of 5 μ M Bay11-7082 for 8 h. Caspase 8 and 3/7 activities were assessed with Caspase-Glo assay kits. Results are relative to control non-treated cells. a – d results are the mean of three independent experiments±S.E.M. ‘ *** ' indicates statistical difference with P
Figure Legend Snippet: MB cell lines displayed different sensitivity to etoposide involving both caspase-dependent and -independent cell death. ( a ) Cell viability was analysed by MTS assay at indicated time points, upon 20 μ M etoposide treatment. ( b ) Viability of D283-MED cells treated with 20 μ M etoposide in the presence of 5 μ M Bay11-7082, 10 μ M Wedelolactone or 5 μ M JSH23 was assessed by MTS assay. ( c ) D283-Med cells were transfected with 100 nM of siRNA directed against p65 or a control scrambled siRNA. After 48 h, cells were treated with 20 μ M etoposide for 8 h and viability was assessed by MTS assay. The knockdown efficiency was controlled by western blot (insert). ( d ) Several MB cell lines were treated with 20 μ M etoposide for indicated times. Caspase 3/7 activity was measured with Caspase-Glo 3/7 assay kit. Results are expressed as fold-induction compared with control non-treated cells as a function of time. ( e ) D283-MED cells were treated with 20 μ M etoposide in presence or absence of 5 μ M Bay11-7082 for 8 h. Caspase 8 and 3/7 activities were assessed with Caspase-Glo assay kits. Results are relative to control non-treated cells. a – d results are the mean of three independent experiments±S.E.M. ‘ *** ' indicates statistical difference with P

Techniques Used: MTS Assay, Transfection, Western Blot, Activity Assay, Caspase-Glo Assay

Etoposide-induced p65 activation is p53 dependent in MB cells. ( a ) Cells were treated with 20 μ M etoposide for 6 h and p53 expression levels were measured by western blot in different MB cell lines. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( b ) qPCR relative quantification of mdm2 mRNA expression in all cell lines was assessed upon 8 h of etoposide treatment (20 μ M). The plot represents the relative fold induction compared to control non-treated cells. ( c – e ) D283-MED cells were transfected with 100 nM of siRNA directed against p53 or a control-scrambled siRNA for 48 h before etoposide treatment. The efficiency of the knockdown is shown on the blot in panel c. ( c ) Cells were treated with 20 μ M etoposide for 6 h. p65 phosphorylation levels were analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( d ) Cells were treated with 20 μ M etoposide for 8 h and viability was assessed using MTS assay. ( e ) Cells were treated with etoposide (20 μ M, 8 h) and caspase activity was measured using Caspase-Glo 8 and 3/7 assay kits. Results are relative to control non-treated cells
Figure Legend Snippet: Etoposide-induced p65 activation is p53 dependent in MB cells. ( a ) Cells were treated with 20 μ M etoposide for 6 h and p53 expression levels were measured by western blot in different MB cell lines. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( b ) qPCR relative quantification of mdm2 mRNA expression in all cell lines was assessed upon 8 h of etoposide treatment (20 μ M). The plot represents the relative fold induction compared to control non-treated cells. ( c – e ) D283-MED cells were transfected with 100 nM of siRNA directed against p53 or a control-scrambled siRNA for 48 h before etoposide treatment. The efficiency of the knockdown is shown on the blot in panel c. ( c ) Cells were treated with 20 μ M etoposide for 6 h. p65 phosphorylation levels were analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( d ) Cells were treated with 20 μ M etoposide for 8 h and viability was assessed using MTS assay. ( e ) Cells were treated with etoposide (20 μ M, 8 h) and caspase activity was measured using Caspase-Glo 8 and 3/7 assay kits. Results are relative to control non-treated cells

Techniques Used: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, MTS Assay, Activity Assay

Etoposide induces a delayed p65 activation in some MB cells. ( a ) Four MB cell lines were treated with 20 μ M etoposide for 6 h. p65 and p65-Ser536 phosphorylation levels were assessed by western blot. The blot presented is representative of three independent experiments. Quantification of the bands was plotted as p65 phosphorylation fold increase compared with the control non-treated cells. ( b ) After 24 h transfection with NF-luc plasmid, cells were treated with 20 μ M etoposide for 8 h before assessment of the luciferase activity. Relative luminescence measurements to t0 are plotted for each cell line. ( c ) D283-MED cells were simultaneously treated for 6 h with 20 μ M etoposide and 5 μ M Bay11-7082 or 10 μ M BMS-345541. p65 phosphorylation was analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( d ) D283-MED cells transfected with p65-RedXP and I κ B α -EGFP were treated with 20 μ M etoposide or 10 ng/ml TNF α . Time-lapse confocal microscopy was performed as described in Materials and Methods. Mean fluorescence intensities for individual cells were analysed. A typical cell for each treatment was plotted as the p65 nuclear/cytoplasmic ratio as a function of time. ( e ) D283-MED cells transfected with NF-Luc vector were treated with 20 μ M etoposide or 10 ng/ml TNF α . Real-time luminescence signal was captured as described in Materials and Methods. The graph represents mean luminescence intensities measured in whole field. ( f ) D283-MED cells were pretreated 30 min with 5 μ g/ml of the transcription inhibitor actinomycin-D, prior to a 10 min treatment with 10 ng/ml TNF α , or a 4 h treatment with 20 μ M etoposide. p65 phosphorylation levels were analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown
Figure Legend Snippet: Etoposide induces a delayed p65 activation in some MB cells. ( a ) Four MB cell lines were treated with 20 μ M etoposide for 6 h. p65 and p65-Ser536 phosphorylation levels were assessed by western blot. The blot presented is representative of three independent experiments. Quantification of the bands was plotted as p65 phosphorylation fold increase compared with the control non-treated cells. ( b ) After 24 h transfection with NF-luc plasmid, cells were treated with 20 μ M etoposide for 8 h before assessment of the luciferase activity. Relative luminescence measurements to t0 are plotted for each cell line. ( c ) D283-MED cells were simultaneously treated for 6 h with 20 μ M etoposide and 5 μ M Bay11-7082 or 10 μ M BMS-345541. p65 phosphorylation was analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown. ( d ) D283-MED cells transfected with p65-RedXP and I κ B α -EGFP were treated with 20 μ M etoposide or 10 ng/ml TNF α . Time-lapse confocal microscopy was performed as described in Materials and Methods. Mean fluorescence intensities for individual cells were analysed. A typical cell for each treatment was plotted as the p65 nuclear/cytoplasmic ratio as a function of time. ( e ) D283-MED cells transfected with NF-Luc vector were treated with 20 μ M etoposide or 10 ng/ml TNF α . Real-time luminescence signal was captured as described in Materials and Methods. The graph represents mean luminescence intensities measured in whole field. ( f ) D283-MED cells were pretreated 30 min with 5 μ g/ml of the transcription inhibitor actinomycin-D, prior to a 10 min treatment with 10 ng/ml TNF α , or a 4 h treatment with 20 μ M etoposide. p65 phosphorylation levels were analysed by western blot. The blot presented is representative of three independent experiments and the plot represents the quantification of the blot shown

Techniques Used: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Confocal Microscopy, Fluorescence

Molecular mechanisms of etoposide-induced cell death in brain tumours. The schematic diagram represents a model of intracellular mechanism induced by etoposide in both GM and MB cells. Depending on the genetic background, cells display different sensitivity to etoposide. Through its genotoxic function, etoposide induces p53 activation. p53 activates the transcription of various genes involved in regulation of cell cycle arrest and cell death as Fas receptor and mdm2. Fas receptor expression at the plasma membrane is able to activate p65 in a FasL-independent manner as well as a caspase-dependent apoptotic cell death. p65 enhances apoptotic death by inducing a caspase-independent cell death. Cells displaying this fully efficient crosstalk are very sensitive to etoposide-induced cell death (D283-MED, D458-MED). Conversely, cells impaired in p53 activation are strongly resistant to cell death (MEB-Med8A, D566-MG, T98G). Interestingly, Cells displaying p53 activity but an impaired p65 activation show intermediate resistance (MHH-Med1, U87MG). MB cell lines are in blue and GM cell lines in green. The grey-to-black gradient illustrates the sensitivity to etoposide-induced cell death. The dotted red lines represent the nods in transduction pathways that are blocked in indicated cell lines
Figure Legend Snippet: Molecular mechanisms of etoposide-induced cell death in brain tumours. The schematic diagram represents a model of intracellular mechanism induced by etoposide in both GM and MB cells. Depending on the genetic background, cells display different sensitivity to etoposide. Through its genotoxic function, etoposide induces p53 activation. p53 activates the transcription of various genes involved in regulation of cell cycle arrest and cell death as Fas receptor and mdm2. Fas receptor expression at the plasma membrane is able to activate p65 in a FasL-independent manner as well as a caspase-dependent apoptotic cell death. p65 enhances apoptotic death by inducing a caspase-independent cell death. Cells displaying this fully efficient crosstalk are very sensitive to etoposide-induced cell death (D283-MED, D458-MED). Conversely, cells impaired in p53 activation are strongly resistant to cell death (MEB-Med8A, D566-MG, T98G). Interestingly, Cells displaying p53 activity but an impaired p65 activation show intermediate resistance (MHH-Med1, U87MG). MB cell lines are in blue and GM cell lines in green. The grey-to-black gradient illustrates the sensitivity to etoposide-induced cell death. The dotted red lines represent the nods in transduction pathways that are blocked in indicated cell lines

Techniques Used: Activation Assay, Expressing, Activity Assay, Transduction

36) Product Images from "Apoptosis: A Four-Week Laboratory Investigation for Advanced Molecular and Cellular Biology Students"

Article Title: Apoptosis: A Four-Week Laboratory Investigation for Advanced Molecular and Cellular Biology Students

Journal: Cell Biology Education

doi: 10.1187/cbe.03-06-0027

Student-generated immunoblot showing PARP-1 cleavage in Jurkat cells following exposure to VP-16. Jurkat cells were exposed to 500 μ M VP-16 (VP) or DMSO (C) and incubated at 37°C for the indicated times. Proteins were isolated, resolved by 8% SDS-PAGE, and electroblotted to a nitrocellulose membrane. The membrane was probed with a monoclonal antibody against PARP-1, followed by a goat anti-mouse IgG-HRP conjugate. Bands were visualized by chemiluminescence. The active PARP-1 (116 kD) is present in cell lysates for up to 3 h but, in contrast to uninduced cell lysates, is no longer detected at 6 h following exposure to VP-16. Note that at 6 h after induction, a very faint band at approximately 85 kD (arrow), likely representing the cleaved form of PARP-1, is detected.
Figure Legend Snippet: Student-generated immunoblot showing PARP-1 cleavage in Jurkat cells following exposure to VP-16. Jurkat cells were exposed to 500 μ M VP-16 (VP) or DMSO (C) and incubated at 37°C for the indicated times. Proteins were isolated, resolved by 8% SDS-PAGE, and electroblotted to a nitrocellulose membrane. The membrane was probed with a monoclonal antibody against PARP-1, followed by a goat anti-mouse IgG-HRP conjugate. Bands were visualized by chemiluminescence. The active PARP-1 (116 kD) is present in cell lysates for up to 3 h but, in contrast to uninduced cell lysates, is no longer detected at 6 h following exposure to VP-16. Note that at 6 h after induction, a very faint band at approximately 85 kD (arrow), likely representing the cleaved form of PARP-1, is detected.

Techniques Used: Generated, Incubation, Isolation, SDS Page

Student-generated DNA laddering in HL-60 cells exposed to three apoptosis-inducing agents. HL-60 cells were exposed to either DMSO alone (C), 500 μ M VP-16 (VP), 11 μ M camptothecin (CP), or 4 μ M cycloheximide (CH) for the indicated times. Cells were lysed, and the DNA was isolated by phenol–chloroform extraction followed by ethanol precipitation. The DNA was resolved by electrophoresis through a 1.2% TAE–agarose gel and visualized with ethidium bromide. The relevant sizes of the standard markers (M) are shown as kilobases (kb). DNA laddering is clearly evident within 4 h after exposure to all three inducers. Note, however, that several DNA samples, especially at the 2-h time point, were likely lost during the isolation procedure.
Figure Legend Snippet: Student-generated DNA laddering in HL-60 cells exposed to three apoptosis-inducing agents. HL-60 cells were exposed to either DMSO alone (C), 500 μ M VP-16 (VP), 11 μ M camptothecin (CP), or 4 μ M cycloheximide (CH) for the indicated times. Cells were lysed, and the DNA was isolated by phenol–chloroform extraction followed by ethanol precipitation. The DNA was resolved by electrophoresis through a 1.2% TAE–agarose gel and visualized with ethidium bromide. The relevant sizes of the standard markers (M) are shown as kilobases (kb). DNA laddering is clearly evident within 4 h after exposure to all three inducers. Note, however, that several DNA samples, especially at the 2-h time point, were likely lost during the isolation procedure.

Techniques Used: Generated, DNA Laddering, Isolation, Ethanol Precipitation, Electrophoresis, Agarose Gel Electrophoresis

Phase-contrast microscopy of HL-60 cells showing membrane blebbing after exposure to etoposide. Six hours after students exposed HL-60 cells to VP-16, cells with membrane blebbing were visualized using a phase-contrast microscope and photographed with a digital camera. Original magnifications: B, C, E, and F, 400×; A and D, 1000× (oil immersion).
Figure Legend Snippet: Phase-contrast microscopy of HL-60 cells showing membrane blebbing after exposure to etoposide. Six hours after students exposed HL-60 cells to VP-16, cells with membrane blebbing were visualized using a phase-contrast microscope and photographed with a digital camera. Original magnifications: B, C, E, and F, 400×; A and D, 1000× (oil immersion).

Techniques Used: Microscopy

Immunodetection of PARP-1 cleavage during apoptosis in HL-60 cells exposed to VP-16. HL-60 cells were exposed to 500 μ M VP-16 (VP) or DMSO (C) and incubated at 37°C for the indicated times. Proteins were isolated, resolved by 8% SDS-PAGE, and electroblotted to a nitrocellulose membrane. Each lane contained the total cellular proteins from approximately 4 × 10 5 cells. The membrane was probed with a monoclonal antibody against PARP-1, followed by a goat anti-mouse IgG–HRP conjugate. Bands were visualized by chemiluminescence. The apparent molecular masses of the active and inactive forms of PARP-1 are 116 and 85 kD, respectively.
Figure Legend Snippet: Immunodetection of PARP-1 cleavage during apoptosis in HL-60 cells exposed to VP-16. HL-60 cells were exposed to 500 μ M VP-16 (VP) or DMSO (C) and incubated at 37°C for the indicated times. Proteins were isolated, resolved by 8% SDS-PAGE, and electroblotted to a nitrocellulose membrane. Each lane contained the total cellular proteins from approximately 4 × 10 5 cells. The membrane was probed with a monoclonal antibody against PARP-1, followed by a goat anti-mouse IgG–HRP conjugate. Bands were visualized by chemiluminescence. The apparent molecular masses of the active and inactive forms of PARP-1 are 116 and 85 kD, respectively.

Techniques Used: Immunodetection, Incubation, Isolation, SDS Page

Student photographs of Hoescht-stained cells to study the effects of anchorage dependence on apoptosis. Anchorage-dependent mouse L929 cells were cultured in 24-well dishes with or without paraffin coating to prevent attachment (D and A, respectively). To determine whether paraffin directly induces apoptosis, anchorage-independent HL-60 cells were cultured in the same way (E and B, respectively). Additionally, L929 cells were exposed to VP-16, a known inducer of apoptosis (F), or medium alone (C) without paraffin. In all cases, chromatin condensation was used to indicate apoptosis. Interestingly, no chromatin condensation was evident in L929 cells under any condition even following exposure to VP-16. Original magnification, 400×.
Figure Legend Snippet: Student photographs of Hoescht-stained cells to study the effects of anchorage dependence on apoptosis. Anchorage-dependent mouse L929 cells were cultured in 24-well dishes with or without paraffin coating to prevent attachment (D and A, respectively). To determine whether paraffin directly induces apoptosis, anchorage-independent HL-60 cells were cultured in the same way (E and B, respectively). Additionally, L929 cells were exposed to VP-16, a known inducer of apoptosis (F), or medium alone (C) without paraffin. In all cases, chromatin condensation was used to indicate apoptosis. Interestingly, no chromatin condensation was evident in L929 cells under any condition even following exposure to VP-16. Original magnification, 400×.

Techniques Used: Staining, Cell Culture

DNA laddering in HL-60 cells exposed to VP-16. HL-60 cells were exposed to either 500 μ M VP-16 (VP) or DMSO (C) for the indicated times. Cells were lysed, and the DNA was isolated by phenol–chloroform extraction followed by ethanol precipitation. The DNA was resolved by electrophoresis through a 1.2% TAE–agarose gel and visualized with ethidium bromide. The outermost lanes contain size standards (M); relevant sizes are shown as kilobases (kb). DNA laddering is clearly evident within 4 h after exposure to VP-16. Note that the control DNA at 8 h was likely lost at some point during the isolation procedure, which occasionally occurs when students perform multiple DNA extractions.
Figure Legend Snippet: DNA laddering in HL-60 cells exposed to VP-16. HL-60 cells were exposed to either 500 μ M VP-16 (VP) or DMSO (C) for the indicated times. Cells were lysed, and the DNA was isolated by phenol–chloroform extraction followed by ethanol precipitation. The DNA was resolved by electrophoresis through a 1.2% TAE–agarose gel and visualized with ethidium bromide. The outermost lanes contain size standards (M); relevant sizes are shown as kilobases (kb). DNA laddering is clearly evident within 4 h after exposure to VP-16. Note that the control DNA at 8 h was likely lost at some point during the isolation procedure, which occasionally occurs when students perform multiple DNA extractions.

Techniques Used: DNA Laddering, Isolation, Ethanol Precipitation, Electrophoresis, Agarose Gel Electrophoresis

37) Product Images from "Heterogeneous nuclear ribonucleoprotein (hnRNP) L promotes DNA damage-induced cell apoptosis by enhancing the translation of p53"

Article Title: Heterogeneous nuclear ribonucleoprotein (hnRNP) L promotes DNA damage-induced cell apoptosis by enhancing the translation of p53

Journal: Oncotarget

doi: 10.18632/oncotarget.17003

Reduction of hnRNP L downregulates p53 expression and relieves cell cycle arrest and DNA damage-induced apoptosis of NIH3T3 cells (A) mRNA levels of p53 target genes including p21, Mdm2 and Puma decrease in NIH3T3 cells transfected with hnRNP L siRNA and under etoposide treatment. At 24 h after transfection with control or hnRNP L siRNA, NIH3T3 cells were treated with or without 100 μM etoposide for 12 h. The levels of p21, MdmM2 and Puma mRNAs were analyzed by qRT-PCR and normalized to RPL32 mRNA levels. mRNA levels in control siRNA transfected and non-etoposide treated cells were set as 1. The bars represent the mean±SEM (n=5). (B, C) hnRNP L silencing lowers p53-mediated G2/M arrest and cell death. At 24 h after transfection with control or hnRNP L siRNA, NIH3T3 cells were treated with 50 μM etoposide for the indicated times and stained with DNA dye, propidium iodide (PI). The data were analyzed by flow cytometry. The bars represent the mean±SEM (n=4). (D) Reduction of hnRNP L increases cell viability. Control siRNA or hnRNP L siRNA transfected cells were exposed to 50 μM etoposide for the indicated times and the cell viability was assessed by MTT assay. The graph represents the mean±SEM (n=3). (E) In immortalized fibroblasts from p53/Mdm2 double-knockout mouse, hnRNP L does not affect cell viability. 50 μM etoposide was added to p53/Mdm2 double-knockout mouse fibroblasts transfected with siCon or sihnRNP L and MTT assay was conducted for measurement of cell viability. The graph represents the mean±SEM (n=3). (F) p53 expression is suppressed by knock-down of hnRNP L, which reduces activation and cleavage of caspase 3. Transfected cells were treated with 50 μM etoposide for the indicated times. Knock-down of hnRNP L was confirmed by WB. (G, H) Cell apoptosis was suppressed by hnRNP L knock-down. TUNEL assay was performed in cells transfected with control or hnRNP L siRNA and treated with 100 μM etoposide for 48 h. Nuclei were stained with Hoechst 33342. (G) Representative image from four independent experiments. (H) The diagram shows relative apoptotic cells measured by TUNEL assay. The bars represent the mean±SEM (n=4). More than 700 cells were analyzed in both group. The number of TUNEL-positive cells in control siRNA transfected cells was set as 1. Data information: In (A-E, H) , * P
Figure Legend Snippet: Reduction of hnRNP L downregulates p53 expression and relieves cell cycle arrest and DNA damage-induced apoptosis of NIH3T3 cells (A) mRNA levels of p53 target genes including p21, Mdm2 and Puma decrease in NIH3T3 cells transfected with hnRNP L siRNA and under etoposide treatment. At 24 h after transfection with control or hnRNP L siRNA, NIH3T3 cells were treated with or without 100 μM etoposide for 12 h. The levels of p21, MdmM2 and Puma mRNAs were analyzed by qRT-PCR and normalized to RPL32 mRNA levels. mRNA levels in control siRNA transfected and non-etoposide treated cells were set as 1. The bars represent the mean±SEM (n=5). (B, C) hnRNP L silencing lowers p53-mediated G2/M arrest and cell death. At 24 h after transfection with control or hnRNP L siRNA, NIH3T3 cells were treated with 50 μM etoposide for the indicated times and stained with DNA dye, propidium iodide (PI). The data were analyzed by flow cytometry. The bars represent the mean±SEM (n=4). (D) Reduction of hnRNP L increases cell viability. Control siRNA or hnRNP L siRNA transfected cells were exposed to 50 μM etoposide for the indicated times and the cell viability was assessed by MTT assay. The graph represents the mean±SEM (n=3). (E) In immortalized fibroblasts from p53/Mdm2 double-knockout mouse, hnRNP L does not affect cell viability. 50 μM etoposide was added to p53/Mdm2 double-knockout mouse fibroblasts transfected with siCon or sihnRNP L and MTT assay was conducted for measurement of cell viability. The graph represents the mean±SEM (n=3). (F) p53 expression is suppressed by knock-down of hnRNP L, which reduces activation and cleavage of caspase 3. Transfected cells were treated with 50 μM etoposide for the indicated times. Knock-down of hnRNP L was confirmed by WB. (G, H) Cell apoptosis was suppressed by hnRNP L knock-down. TUNEL assay was performed in cells transfected with control or hnRNP L siRNA and treated with 100 μM etoposide for 48 h. Nuclei were stained with Hoechst 33342. (G) Representative image from four independent experiments. (H) The diagram shows relative apoptotic cells measured by TUNEL assay. The bars represent the mean±SEM (n=4). More than 700 cells were analyzed in both group. The number of TUNEL-positive cells in control siRNA transfected cells was set as 1. Data information: In (A-E, H) , * P

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, MTT Assay, Double Knockout, Activation Assay, Western Blot, TUNEL Assay

hnRNP L controls the expression of p53 through translational regulation (A, B) hnRNP L does not affect endogenous p53 mRNA levels in either normal or DNA-damaged cells. Control siRNA or hnRNP L siRNA was transfected into (A) NIH3T3 and (B) B16F10 and cells treated with 100 μM etoposide for 1 hour and 4 hours, respectively. Endogenous p53 mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR) and normalized to β-actin. The bars represent the mean±SEM (n=3). (C, D) Knock-down of hnRNP L does not affect p53 protein stability. After 100 μM etoposide was added to (C) NIH3T3 and (D) B16F10 cells, 50 μg/ml cycloheximide (CHX) was then added for the indicated times. Endogenous p53 levels and knock-down of hnRNP L were determined by WB. The amount of p53 protein was normalized to GAPDH. p53 protein levels of 0 time point and control siRNA transfected cells were set as 1. Data show relative p53 protein intensity from four independent experiments (mean±SEM). (E) hnRNP L increases the translation rate of p53 mRNA. After transfection with control or hnRNP L siRNA, 10 μM MG132 was added for the indicated times. Changes in the levels of p53 protein by translation or knock-down were assessed by WB. (F) Metabolic labeling shows that reduction of hnRNP L downregulates protein synthesis of p53. After transfection of NIH3T3 cells with control or hnRNP L siRNA, cells were incubated in medium containing 35 S-labeled methionine ( 35 S-Met) and 35 S-labeled cysteine ( 35 S-Cys) and 10 μM MG132. Newly synthesized p53 proteins were detected after immunoprecipitation (IP) with monoclonal p53 antibody. The numbers at the bottom of the first lane mean the fold increases relative to control. Data information: In (A-D) ; Two-way ANOVA
Figure Legend Snippet: hnRNP L controls the expression of p53 through translational regulation (A, B) hnRNP L does not affect endogenous p53 mRNA levels in either normal or DNA-damaged cells. Control siRNA or hnRNP L siRNA was transfected into (A) NIH3T3 and (B) B16F10 and cells treated with 100 μM etoposide for 1 hour and 4 hours, respectively. Endogenous p53 mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR) and normalized to β-actin. The bars represent the mean±SEM (n=3). (C, D) Knock-down of hnRNP L does not affect p53 protein stability. After 100 μM etoposide was added to (C) NIH3T3 and (D) B16F10 cells, 50 μg/ml cycloheximide (CHX) was then added for the indicated times. Endogenous p53 levels and knock-down of hnRNP L were determined by WB. The amount of p53 protein was normalized to GAPDH. p53 protein levels of 0 time point and control siRNA transfected cells were set as 1. Data show relative p53 protein intensity from four independent experiments (mean±SEM). (E) hnRNP L increases the translation rate of p53 mRNA. After transfection with control or hnRNP L siRNA, 10 μM MG132 was added for the indicated times. Changes in the levels of p53 protein by translation or knock-down were assessed by WB. (F) Metabolic labeling shows that reduction of hnRNP L downregulates protein synthesis of p53. After transfection of NIH3T3 cells with control or hnRNP L siRNA, cells were incubated in medium containing 35 S-labeled methionine ( 35 S-Met) and 35 S-labeled cysteine ( 35 S-Cys) and 10 μM MG132. Newly synthesized p53 proteins were detected after immunoprecipitation (IP) with monoclonal p53 antibody. The numbers at the bottom of the first lane mean the fold increases relative to control. Data information: In (A-D) ; Two-way ANOVA

Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Labeling, Incubation, Synthesized, Immunoprecipitation

hnRNP L enhances IRES activity of p53 5’UTR (A) Schematic representation of the bicistronic luciferase pRF plasmids used for the detection of p53 5’UTR IRES activity. The 157bp p53 5’UTR was inserted between the two cistrons, Renilla luciferase (RLUC) and firefly luciferase (FLUC). (B) IRES activity of p53 5’UTR is increased under hnRNP L overexpression. NIH3T3 cells were transfected with flag Mock or flag hnRNP L and 24 h later with pRF mock vector or pRF p53 5’UTR vector. Luciferase activity is shown as the ratio of FLUC to RLUC and IRES activity of pRF mock and flag Mock transfected cells was set as 1. The bars represent the mean±SEM (n=3). (C, D) Suppression of p53 5’UTR IRES activity is observed after knock-down of hnRNP L. At 24 h after transfection with control or hnRNP L siRNA, pRF mock or pRF mp53 5’UTR vector was transfected into (C) NIH3T3 and (D) B16F10 cells. IRES activity of pRF mock and control siRNA transfected cells was set as 1. The bars represent the mean±SEM (n=7, n=4). (E) Increase of p53 5’UTR IRES activity under etoposide treatment is diminished through the reduction of hnRNP L. Cells were transfected with pRF p53 5’UTR at 24 h after transfection with either control or hnRNP L siRNA, and DMSO or etoposide was added at 18 h after transfection. IRES activity of control siRNA transfected and DMSO treated NIH3T3 cells was set as 1. The bars represent the mean±SEM (n=7). Data information: In (B–E) , n.s., non-significant, * P
Figure Legend Snippet: hnRNP L enhances IRES activity of p53 5’UTR (A) Schematic representation of the bicistronic luciferase pRF plasmids used for the detection of p53 5’UTR IRES activity. The 157bp p53 5’UTR was inserted between the two cistrons, Renilla luciferase (RLUC) and firefly luciferase (FLUC). (B) IRES activity of p53 5’UTR is increased under hnRNP L overexpression. NIH3T3 cells were transfected with flag Mock or flag hnRNP L and 24 h later with pRF mock vector or pRF p53 5’UTR vector. Luciferase activity is shown as the ratio of FLUC to RLUC and IRES activity of pRF mock and flag Mock transfected cells was set as 1. The bars represent the mean±SEM (n=3). (C, D) Suppression of p53 5’UTR IRES activity is observed after knock-down of hnRNP L. At 24 h after transfection with control or hnRNP L siRNA, pRF mock or pRF mp53 5’UTR vector was transfected into (C) NIH3T3 and (D) B16F10 cells. IRES activity of pRF mock and control siRNA transfected cells was set as 1. The bars represent the mean±SEM (n=7, n=4). (E) Increase of p53 5’UTR IRES activity under etoposide treatment is diminished through the reduction of hnRNP L. Cells were transfected with pRF p53 5’UTR at 24 h after transfection with either control or hnRNP L siRNA, and DMSO or etoposide was added at 18 h after transfection. IRES activity of control siRNA transfected and DMSO treated NIH3T3 cells was set as 1. The bars represent the mean±SEM (n=7). Data information: In (B–E) , n.s., non-significant, * P

Techniques Used: Activity Assay, Luciferase, Over Expression, Transfection, Plasmid Preparation

hnRNP L interacts with p53 mRNA and the binding apparently increases after DNA damage (A, B) In vitro binding assays were performed by incubating in vitro transcribed biotin-p53 5’UTR with (A) NIH3T3 or (B) B16F10 cell extracts and followed by pull down with streptavidin beads. The binding between p53 5’UTR and hnRNP L was confirmed by Western blotting. GAPDH was used as negative control. Non-biotinylated p53 5’UTR was used as competitor. (C) hnRNP L directly binds to p53 5’UTR. Purified hnRNP L proteins were incubated with in vitro transcribed biotin-p53 5’UTR. (D) Under DNA damage conditions, the amount of hnRNP L proteins interacting with p53 5’UTR increases. In vitro transcribed biotin-p53 5’UTR was incubated with cytoplasmic extracts of non-treated (Con) or etoposide-treated (Eto) NIH3T3 cells. hnRNP U was used as negative control and GAPDH was used as loading and negative control. The numbers at the bottom mean the fold increases relative to control. (E) Endogenous hnRNP L binds endogenous p53 mRNA and the binding increases under etoposide treatment. Lysates of non-treated (Control) and etoposide treated (Etoposide) NIH3T3 cells were used for RNA-immunoprecipitation (RNAIP) analysis using IgG control and hnRNP L antibody. RNA abundance in IP samples was determined by qRT-PCR. The levels of p53 mRNA were normalized to GAPDH mRNA levels. p53 mRNA level in control-IgG sample was set as 1. The bars represent the mean±SEM (n=3). (F) hnRNP L binds to p53 5’UTR 1-109 region. pRF Mock, pRF p53 5’UTR 1-157 or pRF p53 5’UTR 110-157 vector was transfected into NIH3T3 cells. 24h later, cells were lysed and the lysates were used for RNAIP using control IgG and hnRNP L antibody. RNA abundance was determined by qRT-PCR. The levels of FLUC mRNA in IP samples were normalized to input FLUC mRNA levels. FLUC mRNA level in control IgG sample of the pRF Mock transfected cells was set as 1. The bars represent the mean±SEM (n=4). (G) The region between nucleotides 87 and 109 of p53 5’UTR is important for IRES activity of p53 5’UTR. To confirm IRES activities of serial deletion constructs, luciferase assay was carried out. Luciferase activity is shown as the ratio of FLUC to RLUC. IRES activity of p53 5’UTR 1–157 full length construct was set as 1. The bars represent the mean±SEM (n=3). (H) To identify the binding region of hnRNP L to the 5’UTR of p53 mRNA, in vitro binding assays were conducted. Biotin-labeled p53 5’UTR constructs were incubated with NIH3T3 cell extracts. The interaction of p53 5’UTR and hnRNP L was verified by Western blotting. GAPDH was used as negative control. Data information: In (E-G), * P
Figure Legend Snippet: hnRNP L interacts with p53 mRNA and the binding apparently increases after DNA damage (A, B) In vitro binding assays were performed by incubating in vitro transcribed biotin-p53 5’UTR with (A) NIH3T3 or (B) B16F10 cell extracts and followed by pull down with streptavidin beads. The binding between p53 5’UTR and hnRNP L was confirmed by Western blotting. GAPDH was used as negative control. Non-biotinylated p53 5’UTR was used as competitor. (C) hnRNP L directly binds to p53 5’UTR. Purified hnRNP L proteins were incubated with in vitro transcribed biotin-p53 5’UTR. (D) Under DNA damage conditions, the amount of hnRNP L proteins interacting with p53 5’UTR increases. In vitro transcribed biotin-p53 5’UTR was incubated with cytoplasmic extracts of non-treated (Con) or etoposide-treated (Eto) NIH3T3 cells. hnRNP U was used as negative control and GAPDH was used as loading and negative control. The numbers at the bottom mean the fold increases relative to control. (E) Endogenous hnRNP L binds endogenous p53 mRNA and the binding increases under etoposide treatment. Lysates of non-treated (Control) and etoposide treated (Etoposide) NIH3T3 cells were used for RNA-immunoprecipitation (RNAIP) analysis using IgG control and hnRNP L antibody. RNA abundance in IP samples was determined by qRT-PCR. The levels of p53 mRNA were normalized to GAPDH mRNA levels. p53 mRNA level in control-IgG sample was set as 1. The bars represent the mean±SEM (n=3). (F) hnRNP L binds to p53 5’UTR 1-109 region. pRF Mock, pRF p53 5’UTR 1-157 or pRF p53 5’UTR 110-157 vector was transfected into NIH3T3 cells. 24h later, cells were lysed and the lysates were used for RNAIP using control IgG and hnRNP L antibody. RNA abundance was determined by qRT-PCR. The levels of FLUC mRNA in IP samples were normalized to input FLUC mRNA levels. FLUC mRNA level in control IgG sample of the pRF Mock transfected cells was set as 1. The bars represent the mean±SEM (n=4). (G) The region between nucleotides 87 and 109 of p53 5’UTR is important for IRES activity of p53 5’UTR. To confirm IRES activities of serial deletion constructs, luciferase assay was carried out. Luciferase activity is shown as the ratio of FLUC to RLUC. IRES activity of p53 5’UTR 1–157 full length construct was set as 1. The bars represent the mean±SEM (n=3). (H) To identify the binding region of hnRNP L to the 5’UTR of p53 mRNA, in vitro binding assays were conducted. Biotin-labeled p53 5’UTR constructs were incubated with NIH3T3 cell extracts. The interaction of p53 5’UTR and hnRNP L was verified by Western blotting. GAPDH was used as negative control. Data information: In (E-G), * P

Techniques Used: Binding Assay, In Vitro, Western Blot, Negative Control, Purification, Incubation, Immunoprecipitation, Quantitative RT-PCR, Plasmid Preparation, Transfection, Activity Assay, Construct, Luciferase, Labeling

Accumulation of p53 is enhanced by an increase in hnRNP L after DNA damage-inducing drug treatment (A, B) Endogenous mouse p53 and hnRNP L are induced after etoposide treatment. (A) NIH3T3 and (B) B16F10 cells were treated with 100 μM etoposide for the indicated times. Treatment with dimethylsulfoxide (DMSO) for 10 h was used as vehicle control (Veh). The levels of endogenous proteins were analyzed by western blotting (WB) using anti-p53, anti-GAPDH, anti-hnRNP L and anti-γH2AX antibodies. The housekeeping protein, GAPDH was used as loading control. (C) Protein level of cytosolic hnRNP L increases after treatment with etoposide. NIH3T3 cells were fractionated into cytosol and nucleus after exposure to 100 μM etoposide. hnRNP L protein levels of fractionated cytosolic lysate were determined by WB using anti-hnRNP L antibody. GAPDH protein was used as loading control and cytosol marker. Lamin B protein was analyzed as nucleus marker. DMSO treatment for 4 h was used as vehicle control. Nu, Nuclear lysate. The numbers at the bottom mean the fold increases relative to control. The amount of hnRNP L was normalized to GAPDH. (D, E) hnRNP L overexpression results in increased p53 protein. Flag-tagged hnRNP L was transfected on (D) NIH3T3 and (E) B16F10 cells. Flag-tagged hnRNP L overexpression was confirmed by WB using anti-Flag antibody. (F, G) Induction of p53 is impaired on both (F) NIH3T3 and (G) B16F10 cells by knock-down of hnRNP L after 100 μM etoposide treatment. Knock-down of hnRNP L was confirmed by WB using anti-hnRNP L antibody.
Figure Legend Snippet: Accumulation of p53 is enhanced by an increase in hnRNP L after DNA damage-inducing drug treatment (A, B) Endogenous mouse p53 and hnRNP L are induced after etoposide treatment. (A) NIH3T3 and (B) B16F10 cells were treated with 100 μM etoposide for the indicated times. Treatment with dimethylsulfoxide (DMSO) for 10 h was used as vehicle control (Veh). The levels of endogenous proteins were analyzed by western blotting (WB) using anti-p53, anti-GAPDH, anti-hnRNP L and anti-γH2AX antibodies. The housekeeping protein, GAPDH was used as loading control. (C) Protein level of cytosolic hnRNP L increases after treatment with etoposide. NIH3T3 cells were fractionated into cytosol and nucleus after exposure to 100 μM etoposide. hnRNP L protein levels of fractionated cytosolic lysate were determined by WB using anti-hnRNP L antibody. GAPDH protein was used as loading control and cytosol marker. Lamin B protein was analyzed as nucleus marker. DMSO treatment for 4 h was used as vehicle control. Nu, Nuclear lysate. The numbers at the bottom mean the fold increases relative to control. The amount of hnRNP L was normalized to GAPDH. (D, E) hnRNP L overexpression results in increased p53 protein. Flag-tagged hnRNP L was transfected on (D) NIH3T3 and (E) B16F10 cells. Flag-tagged hnRNP L overexpression was confirmed by WB using anti-Flag antibody. (F, G) Induction of p53 is impaired on both (F) NIH3T3 and (G) B16F10 cells by knock-down of hnRNP L after 100 μM etoposide treatment. Knock-down of hnRNP L was confirmed by WB using anti-hnRNP L antibody.

Techniques Used: Western Blot, Marker, Over Expression, Transfection

38) Product Images from "Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells"

Article Title: Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells

Journal: Scientific Reports

doi: 10.1038/srep34064

Etoposide induced cell swelling and cytotoxicity through ROS generation. HK-2 cells were pre-treated with NAC (5 mM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( A ) Cell morphology was observed using phase contrast microscopy (original magnification X100 ). ( B ) The protein expression levels of cleaved-caspase3 detected by western blot were quantified using β-actin as a loading control. ( C ) Analysis of cell death including necrosis, apoptosis and necrosis with apoptosis, as measured using Annexin V and propidium iodide (PI) double staining. Necrotic and/or apoptotic cells were detected by FACS analysis. Histogram indicates the percentage of fluorescence positive cells. ( D ) HK-2 cells were pre-treated with NAC (5 mM) and catalase (100 U/ml) for 1 hour and then treated with etoposide (50 μM) for 48 hours. Cell viability was measured using MTT assay. Data shown represent the mean ± SEM of three independent experiments (*P
Figure Legend Snippet: Etoposide induced cell swelling and cytotoxicity through ROS generation. HK-2 cells were pre-treated with NAC (5 mM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( A ) Cell morphology was observed using phase contrast microscopy (original magnification X100 ). ( B ) The protein expression levels of cleaved-caspase3 detected by western blot were quantified using β-actin as a loading control. ( C ) Analysis of cell death including necrosis, apoptosis and necrosis with apoptosis, as measured using Annexin V and propidium iodide (PI) double staining. Necrotic and/or apoptotic cells were detected by FACS analysis. Histogram indicates the percentage of fluorescence positive cells. ( D ) HK-2 cells were pre-treated with NAC (5 mM) and catalase (100 U/ml) for 1 hour and then treated with etoposide (50 μM) for 48 hours. Cell viability was measured using MTT assay. Data shown represent the mean ± SEM of three independent experiments (*P

Techniques Used: Microscopy, Expressing, Western Blot, Double Staining, FACS, Fluorescence, MTT Assay

Etoposide induced DNA damage and mitochondrial biogenesis through ROS generation. HK-2 cells were pre-treated with NAC (5 mM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( A , B ) Generation of ROS in cytosol ( A ) and mitochondria ( B ) were measured by DCF-DA and MitoSOX™ fluorescence intensity of cells using a FACS system, respectively. ( C ) The protein expression levels of PARP1, C-PARP1, p-ERK Thr202/Tyr204 , ERK, and ATF3 were measured using western blot. β-actin was used as a loading control. ( D ) Mitochondrial mass and respiration were determined by MitoTracker Green FM and MitoTracker Red CMXRos, respectively, using the FACS system. ( E ) Mitochondrial DNA copy number was measured in mitochondrial-encoded genes, such as ND1 and ND4, using RT-PCR and normalized to GAPDH copy number. ( F ) The cytosolic ATP level was measured by CellTiter-Glo ® Luminescent Cell Viability Assay using a luminescence microplate reader; luminescence intensity is presented in the image. ( G–I ) The expression levels of mitochondrial biogenesis-related genes, such as TFAM, PGC-1α , and PGC-1β , were measured using RT-PCR and normalized to GAPDH . All data shown represent the mean ± SEM of three independent experiments (*P
Figure Legend Snippet: Etoposide induced DNA damage and mitochondrial biogenesis through ROS generation. HK-2 cells were pre-treated with NAC (5 mM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( A , B ) Generation of ROS in cytosol ( A ) and mitochondria ( B ) were measured by DCF-DA and MitoSOX™ fluorescence intensity of cells using a FACS system, respectively. ( C ) The protein expression levels of PARP1, C-PARP1, p-ERK Thr202/Tyr204 , ERK, and ATF3 were measured using western blot. β-actin was used as a loading control. ( D ) Mitochondrial mass and respiration were determined by MitoTracker Green FM and MitoTracker Red CMXRos, respectively, using the FACS system. ( E ) Mitochondrial DNA copy number was measured in mitochondrial-encoded genes, such as ND1 and ND4, using RT-PCR and normalized to GAPDH copy number. ( F ) The cytosolic ATP level was measured by CellTiter-Glo ® Luminescent Cell Viability Assay using a luminescence microplate reader; luminescence intensity is presented in the image. ( G–I ) The expression levels of mitochondrial biogenesis-related genes, such as TFAM, PGC-1α , and PGC-1β , were measured using RT-PCR and normalized to GAPDH . All data shown represent the mean ± SEM of three independent experiments (*P

Techniques Used: Fluorescence, FACS, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Viability Assay, Pyrolysis Gas Chromatography

Etoposide induced nuclear envelope ruptures through ERK activation. HK-2 cells were pre-treated with U0126 (20 μM) or FR180204 (10 μM) for 1 hour followed by treatment with etoposide (50 μM) for 48 hours. Nuclei were extracted from the cells using NE-PER ® nuclear and cytoplasmic extraction reagents. ( A ) Nuclear morphological changes were measured using a hematology analyzer and detected by microscopy (original magnification X100 ). ( B ) Nuclei were extracted, seeded and attached on culture dish for 15 minutes, where morphological changes such as nucleus swelling were measured using microscopy (original magnification X100 ). ( C ) Topography of NE was measured using an AFM probe system. Images shown are representative of 3-D topography at 30, 10, and 2 μm scales. ( D ) Nuclear extracts were seeded on coverslips for 15 min and stained with nucleus targeting dyes such as Hoechst 33258 (blue color) and propidium iodide (red color) that were detected using confocal microscopy. Arrows indicate the DNA leakage.
Figure Legend Snippet: Etoposide induced nuclear envelope ruptures through ERK activation. HK-2 cells were pre-treated with U0126 (20 μM) or FR180204 (10 μM) for 1 hour followed by treatment with etoposide (50 μM) for 48 hours. Nuclei were extracted from the cells using NE-PER ® nuclear and cytoplasmic extraction reagents. ( A ) Nuclear morphological changes were measured using a hematology analyzer and detected by microscopy (original magnification X100 ). ( B ) Nuclei were extracted, seeded and attached on culture dish for 15 minutes, where morphological changes such as nucleus swelling were measured using microscopy (original magnification X100 ). ( C ) Topography of NE was measured using an AFM probe system. Images shown are representative of 3-D topography at 30, 10, and 2 μm scales. ( D ) Nuclear extracts were seeded on coverslips for 15 min and stained with nucleus targeting dyes such as Hoechst 33258 (blue color) and propidium iodide (red color) that were detected using confocal microscopy. Arrows indicate the DNA leakage.

Techniques Used: Activation Assay, Microscopy, Staining, Confocal Microscopy

Etoposide induced DNA damage and cytotoxicity through ERK activation. ( A ) HK-2 cells were pre-treated for 1 hour with different MAPK inhibitors (10 μM of SB203580, 20 μM of SP600125 and 20 μM of U0126), and then treated with DMSO (vehicle control) or 50 μM of etoposide. The protein expression levels of γ-H2AX ser139 , PARP1, C-PARP1 and ATF3 detected by western blots. β-actin was used as a loading control. HK-2 cells were pre-treated with U0126 (20 μM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( B ) DNA content in different cell cycle stages was determined by propidium iodide (PI) staining using FACS analysis. This method gives the percentage of cells in each phase as shown in the histogram. ( C ) The protein expression levels of γ-H2AX ser139 , PARP1, C-PARP1, p-ERK Thr202/Tyr204 , ERK, cleaved-lamin A/C, and cleaved-caspase3 detected by western blot. β-actin was used as a loading control. ( D ) Caspase 3/7 activity was measured in a Caspase-Glo 3/7 assay using a luminescence microplate reader; luminescence intensity is shown in the image. ( E ) Proliferation of HK-2 cells treated with either U0126, or etoposide or etoposide in combination with U0126 was measured using the xCELLigence system in real-time at 1 hour intervals for 48 hours. HK-2 cells were seeded on E-Plate 16 devices and grown overnight to form monolayers. The next day, cells were pre-treated with U0126 (20 μM) for 1 hour followed by treatment with etoposide (50 μM). Cell proliferation was measured by changes in electrical impedance (cell index) on the surface. ( F ) Histogram of cell proliferation measured by cell index at 48 hours. ( G ) HK-2 cells were pre-treated for 1 hour with U0126 (20 μM) and FR180204 (10 μM), and then treated with 50 μM of etoposide. Cell viability was measured using MTT assay. ( H ) Cytotoxicity was analyzed using LDH assay. Data shown represent the mean ± SEM of three independent experiments (*P
Figure Legend Snippet: Etoposide induced DNA damage and cytotoxicity through ERK activation. ( A ) HK-2 cells were pre-treated for 1 hour with different MAPK inhibitors (10 μM of SB203580, 20 μM of SP600125 and 20 μM of U0126), and then treated with DMSO (vehicle control) or 50 μM of etoposide. The protein expression levels of γ-H2AX ser139 , PARP1, C-PARP1 and ATF3 detected by western blots. β-actin was used as a loading control. HK-2 cells were pre-treated with U0126 (20 μM) for 1 hour and then treated with etoposide (50 μM) for 48 hours. ( B ) DNA content in different cell cycle stages was determined by propidium iodide (PI) staining using FACS analysis. This method gives the percentage of cells in each phase as shown in the histogram. ( C ) The protein expression levels of γ-H2AX ser139 , PARP1, C-PARP1, p-ERK Thr202/Tyr204 , ERK, cleaved-lamin A/C, and cleaved-caspase3 detected by western blot. β-actin was used as a loading control. ( D ) Caspase 3/7 activity was measured in a Caspase-Glo 3/7 assay using a luminescence microplate reader; luminescence intensity is shown in the image. ( E ) Proliferation of HK-2 cells treated with either U0126, or etoposide or etoposide in combination with U0126 was measured using the xCELLigence system in real-time at 1 hour intervals for 48 hours. HK-2 cells were seeded on E-Plate 16 devices and grown overnight to form monolayers. The next day, cells were pre-treated with U0126 (20 μM) for 1 hour followed by treatment with etoposide (50 μM). Cell proliferation was measured by changes in electrical impedance (cell index) on the surface. ( F ) Histogram of cell proliferation measured by cell index at 48 hours. ( G ) HK-2 cells were pre-treated for 1 hour with U0126 (20 μM) and FR180204 (10 μM), and then treated with 50 μM of etoposide. Cell viability was measured using MTT assay. ( H ) Cytotoxicity was analyzed using LDH assay. Data shown represent the mean ± SEM of three independent experiments (*P

Techniques Used: Activation Assay, Expressing, Western Blot, Staining, FACS, Activity Assay, Caspase-Glo Assay, MTT Assay, Lactate Dehydrogenase Assay

Overview of the mechanism underlying ROS- and ERK-mediated cytotoxicity. Etoposide treatment triggers ROS generation and ERK activation in HK-2 cells. ROS promotes mitochondrial biogenesis and cytosolic ATP induction, which eventually enhance necrosis, but not apoptosis. Whereas, ERK activation causes caspase 3/7 activation that in turn ruptures the nuclear envelope, which eventually induces apoptosis. Furthermore, ERK activation is independent of ROS generation.
Figure Legend Snippet: Overview of the mechanism underlying ROS- and ERK-mediated cytotoxicity. Etoposide treatment triggers ROS generation and ERK activation in HK-2 cells. ROS promotes mitochondrial biogenesis and cytosolic ATP induction, which eventually enhance necrosis, but not apoptosis. Whereas, ERK activation causes caspase 3/7 activation that in turn ruptures the nuclear envelope, which eventually induces apoptosis. Furthermore, ERK activation is independent of ROS generation.

Techniques Used: Activation Assay

Etoposide induced DNA damage, MAPKs activation, and nuclei swelling. HK-2 cells were treated with Etoposide (50 μM) for 48 hours. ( A ) Cell viability was measured using an MTT assay. ( B ) Cell size was measured by forward scattered light (FSC) unit and detected using a FACS system. ( C ) The nucleus area was measured by Hochest33258 staining, and at least 3,000 cells were detected using Cellomics ArrayScan VTI HCS Reader. ( D ) The expression of γ-H2AX ser139 and PARP1 in the nuclei from at least 3,000 cells was measured by immunofluorescence staining, and detected using Cellomics ArrayScan VTI HCS Reader system. ( E ) The nuclear swelling was observed by Hoechst 33258 staining (blue color) and expression of γ-H2AX ser139 (red) and PARP1 (green) in the nuclei was determined by immunofluorescence staining and detected using confocal microscopy (scale bar represents 10 μm). ( F ) The protein expression levels of PARP1, C-PARP1, ATF3, p-ERK Thr202/Tyr204 , ERK, p-p38 Thr180/Tyr182 , p38, p-JNK Thr183/Tyr185 and JNK in etoposide (50 μM) treated cells over time were analyzed by western blot. β-actin was used as a loading control. All data shown represent the mean ± SEM of three independent experiments (*P
Figure Legend Snippet: Etoposide induced DNA damage, MAPKs activation, and nuclei swelling. HK-2 cells were treated with Etoposide (50 μM) for 48 hours. ( A ) Cell viability was measured using an MTT assay. ( B ) Cell size was measured by forward scattered light (FSC) unit and detected using a FACS system. ( C ) The nucleus area was measured by Hochest33258 staining, and at least 3,000 cells were detected using Cellomics ArrayScan VTI HCS Reader. ( D ) The expression of γ-H2AX ser139 and PARP1 in the nuclei from at least 3,000 cells was measured by immunofluorescence staining, and detected using Cellomics ArrayScan VTI HCS Reader system. ( E ) The nuclear swelling was observed by Hoechst 33258 staining (blue color) and expression of γ-H2AX ser139 (red) and PARP1 (green) in the nuclei was determined by immunofluorescence staining and detected using confocal microscopy (scale bar represents 10 μm). ( F ) The protein expression levels of PARP1, C-PARP1, ATF3, p-ERK Thr202/Tyr204 , ERK, p-p38 Thr180/Tyr182 , p38, p-JNK Thr183/Tyr185 and JNK in etoposide (50 μM) treated cells over time were analyzed by western blot. β-actin was used as a loading control. All data shown represent the mean ± SEM of three independent experiments (*P

Techniques Used: Activation Assay, MTT Assay, FACS, Staining, Expressing, Immunofluorescence, Confocal Microscopy, Western Blot

39) Product Images from "TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In VivoTdp2: A Means to Fixing the Ends"

Article Title: TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In VivoTdp2: A Means to Fixing the Ends

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003226

The absence of TDP2 increases etoposide-induced genome instability in vivo . Percentage of micronucleated polychromatic erythrocytes (MN-PCE) among the total number of polychromatic erythrocytes (PCE), examples of which are shown (right), in bone marrow smears of wild-type and Tdp2 Δ1–3 mice 24 h after intraperitoneal injection of 1 mg/kg etoposide or vehicle (10% DMSO). Average ± s.e.m. of 4 (DMSO) and 6 (VP16) animals and statistical significance by paired T test is shown.
Figure Legend Snippet: The absence of TDP2 increases etoposide-induced genome instability in vivo . Percentage of micronucleated polychromatic erythrocytes (MN-PCE) among the total number of polychromatic erythrocytes (PCE), examples of which are shown (right), in bone marrow smears of wild-type and Tdp2 Δ1–3 mice 24 h after intraperitoneal injection of 1 mg/kg etoposide or vehicle (10% DMSO). Average ± s.e.m. of 4 (DMSO) and 6 (VP16) animals and statistical significance by paired T test is shown.

Techniques Used: In Vivo, Mouse Assay, Injection

TDP2 promotes survival following TOP2-induced DSBs. A. Clonogenic survival of the indicated DT40 cell line; wild-type, TDP2 −/−/− and TDP2 −/−/− complemented with human TDP2 (hTDP2) and catalytic-dead human TDP2 (hTDP2 262A) or empty vector (Empty V); following continuous treatment with the indicated concentrations of doxorubicin (left) or mAMSA (right). Average ± s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose when compared to TDP2 −/−/− cells by Two-way ANOVA with Bonferroni post-test is shown. B. Scheme showing the strategy for targeted deletion of the first three exons of Tdp2 in mouse. The wild-type ( Tdp2 + ), conditional ( Tdp2 flEx1–3,neo ) and deleted ( Tdp2 flΔ ) alleles are depicted. The EcoRI-EcoRI fragment of Tdp2 was used in the targeting construct. Southern-blot analysis of PstI -digested DNA from wild-type ( +/+ ), heterozygous ( +/flΔ ) and knock-out ( flΔ/flΔ , from now on denoted Tdp2 Δ1–3 ) mice, using the indicated probe (red line), is shown (bottom right). C. Clonogenic survival of wild-type and Tdp2 Δ1–3 transformed MEFs after 3 h acute exposure to the indicated concentrations of etoposide (left) or the indicated dose of γ-irradiation (right). Average ± s.e.m. of three independent experiments and statistical significance by Two-way ANOVA test with Bonferroni post-test is shown. In all figures (*P≤0.05; **P≤0.01; ***P≤0.005).
Figure Legend Snippet: TDP2 promotes survival following TOP2-induced DSBs. A. Clonogenic survival of the indicated DT40 cell line; wild-type, TDP2 −/−/− and TDP2 −/−/− complemented with human TDP2 (hTDP2) and catalytic-dead human TDP2 (hTDP2 262A) or empty vector (Empty V); following continuous treatment with the indicated concentrations of doxorubicin (left) or mAMSA (right). Average ± s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose when compared to TDP2 −/−/− cells by Two-way ANOVA with Bonferroni post-test is shown. B. Scheme showing the strategy for targeted deletion of the first three exons of Tdp2 in mouse. The wild-type ( Tdp2 + ), conditional ( Tdp2 flEx1–3,neo ) and deleted ( Tdp2 flΔ ) alleles are depicted. The EcoRI-EcoRI fragment of Tdp2 was used in the targeting construct. Southern-blot analysis of PstI -digested DNA from wild-type ( +/+ ), heterozygous ( +/flΔ ) and knock-out ( flΔ/flΔ , from now on denoted Tdp2 Δ1–3 ) mice, using the indicated probe (red line), is shown (bottom right). C. Clonogenic survival of wild-type and Tdp2 Δ1–3 transformed MEFs after 3 h acute exposure to the indicated concentrations of etoposide (left) or the indicated dose of γ-irradiation (right). Average ± s.e.m. of three independent experiments and statistical significance by Two-way ANOVA test with Bonferroni post-test is shown. In all figures (*P≤0.05; **P≤0.01; ***P≤0.005).

Techniques Used: Plasmid Preparation, Construct, Southern Blot, Knock-Out, Mouse Assay, Transformation Assay, Irradiation

TDP2 promotes repair of TOP2-induced DSBs by NHEJ. A. Clonogenic survival of wild-type, TDP2 −/−/− , KU70 −/− and TDP2 −/−/− KU70 −/− DT40 cells following continuous treatment with the indicated concentrations of etoposide. Average ± s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose by Two-way ANOVA with Bonferroni post-test is shown. B. Clonogenic survival of wild-type and BRCA2-mutant human transformed fibroblasts with (Tdp2si) and without (control) TDP2 depletion following 3 h acute exposure to the indicated concentrations of etoposide. Western blot analysis of TDP2 levels in wild type and BRCA2-mutant cell extracts after 48 h of transfection is indicated (inset). Other details as in “A”. C. γH2AX foci induction after 30 min 20 µM etoposide treatment and repair at different times following drug removal in confluency arrested Tdp2 +/+ and Tdp2 Δ1–3 primary MEFs. Representative images of the 3 h repair time point including DAPI counterstain (right) and average ± s.e.m. of at least three independent experiments (left) are shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated. D. G2 primary MEFs (see Matherials and Methods) following 30 min 10 µM etoposide treatment. Other details as in “C”. E. Confluency arrested primary MEFs exposed to 2Gy γ-irradiation. Other details as in “C”.
Figure Legend Snippet: TDP2 promotes repair of TOP2-induced DSBs by NHEJ. A. Clonogenic survival of wild-type, TDP2 −/−/− , KU70 −/− and TDP2 −/−/− KU70 −/− DT40 cells following continuous treatment with the indicated concentrations of etoposide. Average ± s.e.m. of at least three independent experiments and statistical significance at the highest indicated dose by Two-way ANOVA with Bonferroni post-test is shown. B. Clonogenic survival of wild-type and BRCA2-mutant human transformed fibroblasts with (Tdp2si) and without (control) TDP2 depletion following 3 h acute exposure to the indicated concentrations of etoposide. Western blot analysis of TDP2 levels in wild type and BRCA2-mutant cell extracts after 48 h of transfection is indicated (inset). Other details as in “A”. C. γH2AX foci induction after 30 min 20 µM etoposide treatment and repair at different times following drug removal in confluency arrested Tdp2 +/+ and Tdp2 Δ1–3 primary MEFs. Representative images of the 3 h repair time point including DAPI counterstain (right) and average ± s.e.m. of at least three independent experiments (left) are shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated. D. G2 primary MEFs (see Matherials and Methods) following 30 min 10 µM etoposide treatment. Other details as in “C”. E. Confluency arrested primary MEFs exposed to 2Gy γ-irradiation. Other details as in “C”.

Techniques Used: Non-Homologous End Joining, Mutagenesis, Transformation Assay, Western Blot, Transfection, Irradiation

The absence of TDP2 increases etoposide induced homologous recombination. A. Total number of foci per RAD51 foci-containing cell in Tdp2 +/+ and Tdp2 Δ1–3 primary MEFs following 30 min 10 µM etoposide treatment and 2 h recovery (left). Replicating cells were excluded from the analysis. A representative image is shown (right). Average ± s.e.m. from 3 independent experiments and statistical significance by T test is indicated. B. Sister chromatid exchanges (SCEs) scored in Tdp2 +/+ and Tdp2 Δ1–3 transformed MEFs after 30 min acute treatment with the indicated concentration of etoposide. Plots show the number of SCEs per chromosome from individual metaphase spreads (n≥50) obtained in at least two independent experiments. Average ± s.e.m. and statistical significance by Mann-Whitney test is also indicated.
Figure Legend Snippet: The absence of TDP2 increases etoposide induced homologous recombination. A. Total number of foci per RAD51 foci-containing cell in Tdp2 +/+ and Tdp2 Δ1–3 primary MEFs following 30 min 10 µM etoposide treatment and 2 h recovery (left). Replicating cells were excluded from the analysis. A representative image is shown (right). Average ± s.e.m. from 3 independent experiments and statistical significance by T test is indicated. B. Sister chromatid exchanges (SCEs) scored in Tdp2 +/+ and Tdp2 Δ1–3 transformed MEFs after 30 min acute treatment with the indicated concentration of etoposide. Plots show the number of SCEs per chromosome from individual metaphase spreads (n≥50) obtained in at least two independent experiments. Average ± s.e.m. and statistical significance by Mann-Whitney test is also indicated.

Techniques Used: Homologous Recombination, Transformation Assay, Concentration Assay, MANN-WHITNEY

The absence of TDP2 increases etoposide-induced genome instability in mammalian cells. A. Micronuclei (MN, left) and nucleoplasmic bridges (NB, right) in binucleated (following cytochalasin B-mediated cell cycle arrest) Tdp2 +/+ and Tdp2 Δ1–3 transformed MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for representative images. Histogram bars represent the average ± s.e.m. of n≥600 cells coming from three independent experiments. Statistical significance by Mann-Whitney test. B. Primary MEFs in the absence of cytochalasin B treatment (n≥1500). Other details as in “A”. C. Break-type (left) and exchange-type (right) chromosomal aberrations in transformed Tdp2 +/+ and Tdp2 Δ1–3 MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for a representative image. Plots show the number of breaks/exchanges per 100 chromosomes from individual metaphase spreads (n = 100) obtained in at least two independent experiments. Average ± s.e.m. and statistical significance by Mann-Whitney test is also indicated. D. Metaphase spreads from primary MEFs (n = 50). Caffeine was added 4 h after etoposide treatment. Other details as in “C”.
Figure Legend Snippet: The absence of TDP2 increases etoposide-induced genome instability in mammalian cells. A. Micronuclei (MN, left) and nucleoplasmic bridges (NB, right) in binucleated (following cytochalasin B-mediated cell cycle arrest) Tdp2 +/+ and Tdp2 Δ1–3 transformed MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for representative images. Histogram bars represent the average ± s.e.m. of n≥600 cells coming from three independent experiments. Statistical significance by Mann-Whitney test. B. Primary MEFs in the absence of cytochalasin B treatment (n≥1500). Other details as in “A”. C. Break-type (left) and exchange-type (right) chromosomal aberrations in transformed Tdp2 +/+ and Tdp2 Δ1–3 MEFs following acute treatment (30 min) with indicated dose of etoposide. See insets for a representative image. Plots show the number of breaks/exchanges per 100 chromosomes from individual metaphase spreads (n = 100) obtained in at least two independent experiments. Average ± s.e.m. and statistical significance by Mann-Whitney test is also indicated. D. Metaphase spreads from primary MEFs (n = 50). Caffeine was added 4 h after etoposide treatment. Other details as in “C”.

Techniques Used: Transformation Assay, MANN-WHITNEY

The absence of TDP2 causes etoposide sensitivity in vivo . A. 8-week old wild-type and Tdp2 Δ1–3 littermates were intraperitoneally injected with a single 75 mg/kg dose of etoposide or vehicle (DMSO) and body weight was recorded in the following 6 days. Average ± s.e.m. of the percentage of initial body weight from at least 8 mice and statistical significance by One-way ANOVA with Bonferroni post-test is shown. B. Representative image of hematoxylin-eosin stained jejunum slices from wild-type and Tdp2 Δ1–3 animals 6 days after etoposide treatment. C. Macroscopic (left) and histological (right) representative image of spleen and thymus from wild-type and Tdp2 Δ1–3 animals 6 days after treatment. Average weight of these organs ± s.e.m. and statistical significance by Two-way ANOVA with Bonferroni post-test is shown (centre). D. FACS analysis of B-cells in bone marrow (top and bottom-left) and T-cells in thymus (bottom right) in wild-type and Tdp2 Δ1–3 animals 6 days after treatment. See insets to compare etoposide treated samples when required. Average percentage of the indicated cell type among the total number of cells in the corresponding tissue ± s.e.m. of at least 3 animals and statistical significance by Two-way ANOVA with Bonferroni post-test is shown.
Figure Legend Snippet: The absence of TDP2 causes etoposide sensitivity in vivo . A. 8-week old wild-type and Tdp2 Δ1–3 littermates were intraperitoneally injected with a single 75 mg/kg dose of etoposide or vehicle (DMSO) and body weight was recorded in the following 6 days. Average ± s.e.m. of the percentage of initial body weight from at least 8 mice and statistical significance by One-way ANOVA with Bonferroni post-test is shown. B. Representative image of hematoxylin-eosin stained jejunum slices from wild-type and Tdp2 Δ1–3 animals 6 days after etoposide treatment. C. Macroscopic (left) and histological (right) representative image of spleen and thymus from wild-type and Tdp2 Δ1–3 animals 6 days after treatment. Average weight of these organs ± s.e.m. and statistical significance by Two-way ANOVA with Bonferroni post-test is shown (centre). D. FACS analysis of B-cells in bone marrow (top and bottom-left) and T-cells in thymus (bottom right) in wild-type and Tdp2 Δ1–3 animals 6 days after treatment. See insets to compare etoposide treated samples when required. Average percentage of the indicated cell type among the total number of cells in the corresponding tissue ± s.e.m. of at least 3 animals and statistical significance by Two-way ANOVA with Bonferroni post-test is shown.

Techniques Used: In Vivo, Injection, Mouse Assay, Staining, FACS

40) Product Images from "Characterization of stress response in human retinal epithelial cells"

Article Title: Characterization of stress response in human retinal epithelial cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2012.01652.x

Analysis of LEI/L-DNase II in caspase-independent apoptosis. ( A ) Indirect immunofluorescence experiments were performed on ARPE-19 cells untreated or treated with HMA for 24 hrs at the indicated concentrations or with 250 μM etoposide for the indicated times. DAPI (blue) or anti-LEI/L-DNase II antibody (red) was used. Scale bar: 25 μm. ( B ) ARPE-19 cells were treated with 40 μM HMA for 24 hrs and analysed by Western blot using anti-LEI/L-DNase II antibody. β actin was used as loading control. Right panel shows the quantification of the LEI/L-DNase II western. Black bars represent LEI/actin ratio, white bar represents L-DNase II/actin ratio. There is an increase of LEI expression and of L-DNase II in treated cells. Means between treated and untreated cells are statistically different as compared by using t -test ( P
Figure Legend Snippet: Analysis of LEI/L-DNase II in caspase-independent apoptosis. ( A ) Indirect immunofluorescence experiments were performed on ARPE-19 cells untreated or treated with HMA for 24 hrs at the indicated concentrations or with 250 μM etoposide for the indicated times. DAPI (blue) or anti-LEI/L-DNase II antibody (red) was used. Scale bar: 25 μm. ( B ) ARPE-19 cells were treated with 40 μM HMA for 24 hrs and analysed by Western blot using anti-LEI/L-DNase II antibody. β actin was used as loading control. Right panel shows the quantification of the LEI/L-DNase II western. Black bars represent LEI/actin ratio, white bar represents L-DNase II/actin ratio. There is an increase of LEI expression and of L-DNase II in treated cells. Means between treated and untreated cells are statistically different as compared by using t -test ( P

Techniques Used: Immunofluorescence, Western Blot, Expressing

HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P
Figure Legend Snippet: HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P

Techniques Used: Western Blot

Indirect Immunofluorescence analysis of PARP-1 activity and AIF-dependent parthanatos. ( A ) ARPE-19 cells treated with either HMA or etoposide were probed with anti-PAR antibody to evaluate PAR synthesis. ( B ) Evaluation of AIF localization in HMA-treated ARPE-19 cells. Experiments were carried out with anti-mtHSP70 (to label mitochondria, red fluorescence) and anti-AIF (green fluorescence) antibodies. Scale bar 25 μm.
Figure Legend Snippet: Indirect Immunofluorescence analysis of PARP-1 activity and AIF-dependent parthanatos. ( A ) ARPE-19 cells treated with either HMA or etoposide were probed with anti-PAR antibody to evaluate PAR synthesis. ( B ) Evaluation of AIF localization in HMA-treated ARPE-19 cells. Experiments were carried out with anti-mtHSP70 (to label mitochondria, red fluorescence) and anti-AIF (green fluorescence) antibodies. Scale bar 25 μm.

Techniques Used: Immunofluorescence, Activity Assay, Fluorescence

HMA effect on ARPE-19 cell viability and proliferation. ARPE-19 cells were treated with increasing concentrations of HMA (20–120 μM) for up to 72 hrs. Cell incubation with 250 μM etoposide for up to 72 hrs was used as an internal standard for caspase-dependent apoptosis inducers. ( A ) Cell viability was evaluated by the MTT assay after 24, 48 and 72 hrs of treatment. Results are expressed as the mean ± SD of three independent experiments. All measurements were significantly different as calculated using a one way anova test P
Figure Legend Snippet: HMA effect on ARPE-19 cell viability and proliferation. ARPE-19 cells were treated with increasing concentrations of HMA (20–120 μM) for up to 72 hrs. Cell incubation with 250 μM etoposide for up to 72 hrs was used as an internal standard for caspase-dependent apoptosis inducers. ( A ) Cell viability was evaluated by the MTT assay after 24, 48 and 72 hrs of treatment. Results are expressed as the mean ± SD of three independent experiments. All measurements were significantly different as calculated using a one way anova test P

Techniques Used: Incubation, MTT Assay

Analysis of caspase-dependent apoptosis in ARPE-19 cells treated with HMA. ( A ) ARPE-19 cells were treated with different HMA concentrations (40–120 μM) for 24 hrs and then analysed using Western blot. Etoposide (250 μM), administered for 24 hrs, was used as an internal standard. As a positive control for apoptosis, HeLa cells were treated with 100 μM etoposide for 3 hrs followed by 24 hrs of recovery in drug-free medium. The activation of caspases 3, 8 and 9 was investigated. Only caspase 3 was slightly activated in 120 μM HMA treated cells. ( B ) ARPE-19 cells were treated for 24 hrs with HMA (40–120 μM); 250 μM etoposide administered for 72 hrs was used as a pro-apoptotic drug. Long-term cultured (LTC) HeLa cells were used as a positive control for apoptosis. Nuclear DNA was extracted and loaded on a 1.8% agarose gel stained with ethidium bromide. No DNA degradation was visible in untreated cells or in cells treated with HMA 40 or 80 μM. A smear was observed in cells treated with 120 μM HMA and a faint ladder is seen in etoposide-treated ARPE-19 cells. ( C ) Upper panel. Western blot analysis of PARP-1 proteolysis was performed on untreated, HMA- or etoposide-treated ARPE-19 cells. HeLa cells treated with etoposide were used as a positive control. γ-Tubulin was used as a loading control. 113 kD: full length PARP-1; 89 kD: cleaved PARP-1. Lower panel shows a quantification of the expression of full length PARP-1 compared to γ-tubulin. At the used loading of proteins on the gel PARP-1 is not detectable in untreated or etoposide-treated cells, but its expression increases with HMA concentration. All the represented means are different from each other as calculated from a one way anova test ( P
Figure Legend Snippet: Analysis of caspase-dependent apoptosis in ARPE-19 cells treated with HMA. ( A ) ARPE-19 cells were treated with different HMA concentrations (40–120 μM) for 24 hrs and then analysed using Western blot. Etoposide (250 μM), administered for 24 hrs, was used as an internal standard. As a positive control for apoptosis, HeLa cells were treated with 100 μM etoposide for 3 hrs followed by 24 hrs of recovery in drug-free medium. The activation of caspases 3, 8 and 9 was investigated. Only caspase 3 was slightly activated in 120 μM HMA treated cells. ( B ) ARPE-19 cells were treated for 24 hrs with HMA (40–120 μM); 250 μM etoposide administered for 72 hrs was used as a pro-apoptotic drug. Long-term cultured (LTC) HeLa cells were used as a positive control for apoptosis. Nuclear DNA was extracted and loaded on a 1.8% agarose gel stained with ethidium bromide. No DNA degradation was visible in untreated cells or in cells treated with HMA 40 or 80 μM. A smear was observed in cells treated with 120 μM HMA and a faint ladder is seen in etoposide-treated ARPE-19 cells. ( C ) Upper panel. Western blot analysis of PARP-1 proteolysis was performed on untreated, HMA- or etoposide-treated ARPE-19 cells. HeLa cells treated with etoposide were used as a positive control. γ-Tubulin was used as a loading control. 113 kD: full length PARP-1; 89 kD: cleaved PARP-1. Lower panel shows a quantification of the expression of full length PARP-1 compared to γ-tubulin. At the used loading of proteins on the gel PARP-1 is not detectable in untreated or etoposide-treated cells, but its expression increases with HMA concentration. All the represented means are different from each other as calculated from a one way anova test ( P

Techniques Used: Western Blot, Positive Control, Activation Assay, Cell Culture, Agarose Gel Electrophoresis, Staining, Expressing, Concentration Assay

41) Product Images from "Tia1 dependent regulation of mRNA subcellular location and translation controls p53 expression in B cells"

Article Title: Tia1 dependent regulation of mRNA subcellular location and translation controls p53 expression in B cells

Journal: Nature Communications

doi: 10.1038/s41467-017-00454-2

ATM kinase inhibition prevents Tia1 dissociation and p53 mRNA translation after DNA damage induction. a Flow cytometry analysis of p53 protein expression in B cells activated with LPS for 48 h and treated with etoposide (20 μM) for the last 4 h. ATM inhibitor KU55933, Chk1/2 inhibitor AZD7762, Chk2 inhibitor II and p38 inhibitor SB203580 (all at 10 μM) were added to the cells 1 h before treatment with etoposide. b Immunoblot analysis of pSer15-p53, p53, Tia1, Tial1, Gadd45a, p21 and β-actin in LPS-activated B cells incubated with KU55933 prior treatment with etoposide. One of the two independent experiments performed is shown. c Total cellular P53 mRNA abundance measured by RT-qPCR. Data are shown as mean + s.e.m. d Tia1 protein: p53 mRNA co-immunoprecipitation in LPS-activated B cells treated with etoposide in the presence of the ATM inhibitor KU55933. An IgG isotype antibody was used as negative control in these RIP assays. Data are from six independent experiments. 2–3 technical replicates were performed in each experiment. Mann–Whitney test was performed for statistical analysis and P values are shown. e Polysome profile of p53 from PolyRibo-3′ RNA-seq experiments performed in LPS-activated B cells incubated or not with the KU55933 inhibitor prior treatment with etoposide. Data are shown as mean + s.e.m. ( n = 4, post hoc pairwise t -test, *** P
Figure Legend Snippet: ATM kinase inhibition prevents Tia1 dissociation and p53 mRNA translation after DNA damage induction. a Flow cytometry analysis of p53 protein expression in B cells activated with LPS for 48 h and treated with etoposide (20 μM) for the last 4 h. ATM inhibitor KU55933, Chk1/2 inhibitor AZD7762, Chk2 inhibitor II and p38 inhibitor SB203580 (all at 10 μM) were added to the cells 1 h before treatment with etoposide. b Immunoblot analysis of pSer15-p53, p53, Tia1, Tial1, Gadd45a, p21 and β-actin in LPS-activated B cells incubated with KU55933 prior treatment with etoposide. One of the two independent experiments performed is shown. c Total cellular P53 mRNA abundance measured by RT-qPCR. Data are shown as mean + s.e.m. d Tia1 protein: p53 mRNA co-immunoprecipitation in LPS-activated B cells treated with etoposide in the presence of the ATM inhibitor KU55933. An IgG isotype antibody was used as negative control in these RIP assays. Data are from six independent experiments. 2–3 technical replicates were performed in each experiment. Mann–Whitney test was performed for statistical analysis and P values are shown. e Polysome profile of p53 from PolyRibo-3′ RNA-seq experiments performed in LPS-activated B cells incubated or not with the KU55933 inhibitor prior treatment with etoposide. Data are shown as mean + s.e.m. ( n = 4, post hoc pairwise t -test, *** P

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Expressing, Incubation, Quantitative RT-PCR, Immunoprecipitation, Negative Control, MANN-WHITNEY, RNA Sequencing Assay

p53 mRNA is stored within stress granules. a Subcellular localization of p53 mRNA and Tia1 protein in LPS-activated B cells treated or not with etoposide. Fluorescence intensity 3D maps were generated for two regions ( white boxes ) to visualize Tia1 protein and p53 mRNA accumulation in cytoplasmic stress granules. Z axis of 3D maps relates to fluorescence intensity (FI). Representative images shown are from one of the two independent experiments performed in which a minimum of five confocal images were analyzed. Data extended in Supplementary Fig. 5a ( Scale bar = 5 μm). b Tia1 protein: p53 mRNA co-immunoprecipitation in LPS-activated B cells treated or not with etoposide. An IgG isotype antibody was used as negative control in these RIP assays. Data are from two independent experiments. In each experiment, 1–3 experimental replicates were performed per condition (Mann–Whitney test, **** P
Figure Legend Snippet: p53 mRNA is stored within stress granules. a Subcellular localization of p53 mRNA and Tia1 protein in LPS-activated B cells treated or not with etoposide. Fluorescence intensity 3D maps were generated for two regions ( white boxes ) to visualize Tia1 protein and p53 mRNA accumulation in cytoplasmic stress granules. Z axis of 3D maps relates to fluorescence intensity (FI). Representative images shown are from one of the two independent experiments performed in which a minimum of five confocal images were analyzed. Data extended in Supplementary Fig. 5a ( Scale bar = 5 μm). b Tia1 protein: p53 mRNA co-immunoprecipitation in LPS-activated B cells treated or not with etoposide. An IgG isotype antibody was used as negative control in these RIP assays. Data are from two independent experiments. In each experiment, 1–3 experimental replicates were performed per condition (Mann–Whitney test, **** P

Techniques Used: Fluorescence, Generated, Immunoprecipitation, Negative Control, MANN-WHITNEY

Proteasome does not regulate p53 protein synthesis in B cells. a Quantitation by flow cytometry of p53 protein expression in LPS-activated B cells treated with MG132 (10 μM), etoposide (20 μM) or actinomycin D (ActD, 5 μg ml −1 ). Data are from six independent experiments with 2 to 4 biological replicates in each. Mann–Whitney test was performed. b Immunoblot analysis of pSer15-p53, p53 and β-actin in activated B cells treated with Lactacystin (Lact, 1 μM), Nutlin-3 (Nut-3, 10 μM), p53 Activator III RITA (1 μM), SJ172550 (SJ, 1 μM), HBX41108 (HBX, 10 μM) or P005091 (P0, 10 μM) prior induction of DNA damage with etoposide. One of the two independent experiments performed is shown. c Quantization by flow cytometry of p53 expression in B cells treated with different doses of the inhibitors described in b . Data are from six biological replicates collected in two independent experiments. Two-way ANOVA and Bonferroni post-test analysis was performed ( ns non-significant, * P
Figure Legend Snippet: Proteasome does not regulate p53 protein synthesis in B cells. a Quantitation by flow cytometry of p53 protein expression in LPS-activated B cells treated with MG132 (10 μM), etoposide (20 μM) or actinomycin D (ActD, 5 μg ml −1 ). Data are from six independent experiments with 2 to 4 biological replicates in each. Mann–Whitney test was performed. b Immunoblot analysis of pSer15-p53, p53 and β-actin in activated B cells treated with Lactacystin (Lact, 1 μM), Nutlin-3 (Nut-3, 10 μM), p53 Activator III RITA (1 μM), SJ172550 (SJ, 1 μM), HBX41108 (HBX, 10 μM) or P005091 (P0, 10 μM) prior induction of DNA damage with etoposide. One of the two independent experiments performed is shown. c Quantization by flow cytometry of p53 expression in B cells treated with different doses of the inhibitors described in b . Data are from six biological replicates collected in two independent experiments. Two-way ANOVA and Bonferroni post-test analysis was performed ( ns non-significant, * P

Techniques Used: Quantitation Assay, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

DNA damage induces changes in mRNA translation decoupled from total mRNA abundance. a Experimental set up for the study of etoposide- induced changes in total mRNA abundance (mRNAseq) and mRNA translation (Polyribosome profiling followed by 3′end RNA sequencing; PolyRibo-3′ RNA-seq) in B cells. Libraries from four biological replicates were generated for both mRNAseq and PolyRibo-3′ RNA-seq. Changes in total cellular mRNA abundance, changes in ribosome-associated mRNAs and changes in polysome distribution after induction of DNA damage were analyzed using DESeq2 (transcripts collapsed by gene id). b Visualization as MA plots of changes in B-cell transcriptome and B-cell translatome after etoposide treatment (20 μM, 4 h) (LPS = L, LPS + Etoposide = LE). Top panel , MA plot showing the mean expression of mRNA transcripts in B cells vs. changes in total mRNA abundance after treatment with etoposide (mRNAseq). Middle panel , MA plot showing the mean abundance of ribosome- associated mRNAs vs. changes in mRNA translation induced by etoposide (PolyRibo-3′ RNA-seq) ( grey —unchanged mRNAs in both datasets, black —mRNAs that change in cellular mRNA abundance only, red —mRNAs with a translational change only, blue —mRNAs that change at both mRNA abundance and translation). Bottom panel , Venn-diagram with the number of mRNAs that change in abundance, translation or both. c Venn-diagram showing the number of mRNA transcripts with significant changes in monosome, light and heavy polysome fractions (mRNA fraction distribution analysis). d Proportion of differentially expressed (DE) mRNAs, differentially ribosome-associated mRNAs or both that show a significant change in polysome distribution. mRNAs are grouped based on significant changes in abundance in any of the fractions (fractions 4 to 16) or in monosomes only (fractions 4 to 7), in light polysomes (fractions 8 to 10) or in heavy polysomes (fractions 11 to16). e Proportion of genes with a change in polysome distribution (defined change in any of the fractions, monosomes, light polysomes and heavy polysomes) that are differentially expressed (DE, change in total mRNA abundance), differentially associated with ribosomes or show a change in RiboLoad (defined as the proportion of reads in heavy polysomes divided by the total of reads mapped to a given mRNA transcript; [LPS + Etoposide vs. LPS and 0.75
Figure Legend Snippet: DNA damage induces changes in mRNA translation decoupled from total mRNA abundance. a Experimental set up for the study of etoposide- induced changes in total mRNA abundance (mRNAseq) and mRNA translation (Polyribosome profiling followed by 3′end RNA sequencing; PolyRibo-3′ RNA-seq) in B cells. Libraries from four biological replicates were generated for both mRNAseq and PolyRibo-3′ RNA-seq. Changes in total cellular mRNA abundance, changes in ribosome-associated mRNAs and changes in polysome distribution after induction of DNA damage were analyzed using DESeq2 (transcripts collapsed by gene id). b Visualization as MA plots of changes in B-cell transcriptome and B-cell translatome after etoposide treatment (20 μM, 4 h) (LPS = L, LPS + Etoposide = LE). Top panel , MA plot showing the mean expression of mRNA transcripts in B cells vs. changes in total mRNA abundance after treatment with etoposide (mRNAseq). Middle panel , MA plot showing the mean abundance of ribosome- associated mRNAs vs. changes in mRNA translation induced by etoposide (PolyRibo-3′ RNA-seq) ( grey —unchanged mRNAs in both datasets, black —mRNAs that change in cellular mRNA abundance only, red —mRNAs with a translational change only, blue —mRNAs that change at both mRNA abundance and translation). Bottom panel , Venn-diagram with the number of mRNAs that change in abundance, translation or both. c Venn-diagram showing the number of mRNA transcripts with significant changes in monosome, light and heavy polysome fractions (mRNA fraction distribution analysis). d Proportion of differentially expressed (DE) mRNAs, differentially ribosome-associated mRNAs or both that show a significant change in polysome distribution. mRNAs are grouped based on significant changes in abundance in any of the fractions (fractions 4 to 16) or in monosomes only (fractions 4 to 7), in light polysomes (fractions 8 to 10) or in heavy polysomes (fractions 11 to16). e Proportion of genes with a change in polysome distribution (defined change in any of the fractions, monosomes, light polysomes and heavy polysomes) that are differentially expressed (DE, change in total mRNA abundance), differentially associated with ribosomes or show a change in RiboLoad (defined as the proportion of reads in heavy polysomes divided by the total of reads mapped to a given mRNA transcript; [LPS + Etoposide vs. LPS and 0.75

Techniques Used: RNA Sequencing Assay, Generated, Expressing

DNA damage induces mRNA translation of p53 in B cells. a , b Global analysis of changes in mRNA abundance ( a ) and mRNA association with ribosomes ( b ) after treatment with etoposide of LPS-activated B cells. Box plots were generated with data from all genes or from only a selection of highly expressed genes. These genes were selected after calculating the mean number of normalized reads annotated to each gene in our mRNAseq and in our PolyRibo-3′ RNA-seq libraries. Then, windows of expression were generated by adding or subtracting 1000 counts (mRNAseq window in log2 is 11.09
Figure Legend Snippet: DNA damage induces mRNA translation of p53 in B cells. a , b Global analysis of changes in mRNA abundance ( a ) and mRNA association with ribosomes ( b ) after treatment with etoposide of LPS-activated B cells. Box plots were generated with data from all genes or from only a selection of highly expressed genes. These genes were selected after calculating the mean number of normalized reads annotated to each gene in our mRNAseq and in our PolyRibo-3′ RNA-seq libraries. Then, windows of expression were generated by adding or subtracting 1000 counts (mRNAseq window in log2 is 11.09

Techniques Used: Generated, Selection, RNA Sequencing Assay, Expressing

Tia1 mRNA targets are highly responsive to DNA damage. a Proportion of Tia1 mRNA targets compared to non-targeted mRNAs that are differentially expressed in total mRNA abundance, in ribosome-associated or both after B-cell treatment with etoposide. Tia1 mRNA targets were defined as those mRNAs in which > 50 unique cDNA counts were mapped to the 3′UTR in our Tia1 iCLIP experiments. b Proportion of Tia1 mRNA targets with a significant change in any polysome fraction, in monosomes, light or heavy fractions. Tia1 mRNA targets were divided based on the number of unique cDNA counts mapped to the 3′UTR (1+, 10+, 25+or 50+). Tia1 untargeted mRNAs were classified as having none (0) cDNA counts mapped to the 3′UTR. c Distribution analysis of the fold change in ribosome-associated mRNA abundance of differentially expressed Tia1 mRNA targets compared to non-targeted mRNAs. Tia1 mRNA targets were sub-divided as in b . d Gene ontology (GOrilla and REViGO) analysis of Tia1 mRNA targets (10+ cDNA counts mapped to the 3′UTR), which mRNA translation is increased or decreased after B-cell treatment with etoposide (extended in Supplementary Data 5 )
Figure Legend Snippet: Tia1 mRNA targets are highly responsive to DNA damage. a Proportion of Tia1 mRNA targets compared to non-targeted mRNAs that are differentially expressed in total mRNA abundance, in ribosome-associated or both after B-cell treatment with etoposide. Tia1 mRNA targets were defined as those mRNAs in which > 50 unique cDNA counts were mapped to the 3′UTR in our Tia1 iCLIP experiments. b Proportion of Tia1 mRNA targets with a significant change in any polysome fraction, in monosomes, light or heavy fractions. Tia1 mRNA targets were divided based on the number of unique cDNA counts mapped to the 3′UTR (1+, 10+, 25+or 50+). Tia1 untargeted mRNAs were classified as having none (0) cDNA counts mapped to the 3′UTR. c Distribution analysis of the fold change in ribosome-associated mRNA abundance of differentially expressed Tia1 mRNA targets compared to non-targeted mRNAs. Tia1 mRNA targets were sub-divided as in b . d Gene ontology (GOrilla and REViGO) analysis of Tia1 mRNA targets (10+ cDNA counts mapped to the 3′UTR), which mRNA translation is increased or decreased after B-cell treatment with etoposide (extended in Supplementary Data 5 )

Techniques Used:

42) Product Images from "Hypoxia-Induced Decrease in p53 Protein Level and Increase in c-jun DNA Binding Activity Results in Cancer Cell Resistance to Etoposide 1 DNA Binding Activity Results in Cancer Cell Resistance to Etoposide 1 , 2"

Article Title: Hypoxia-Induced Decrease in p53 Protein Level and Increase in c-jun DNA Binding Activity Results in Cancer Cell Resistance to Etoposide 1 DNA Binding Activity Results in Cancer Cell Resistance to Etoposide 1 , 2

Journal: Neoplasia (New York, N.Y.)

doi:

Effect of c-jun silencing on hypoxia-induced resistance to etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM c-jun siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx; 21% O 2 ) or
Figure Legend Snippet: Effect of c-jun silencing on hypoxia-induced resistance to etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM c-jun siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx; 21% O 2 ) or

Techniques Used: Transfection, Negative Control, Incubation

Schematic representation of the hypoxia-induced mechanisms responsible for cancer cell resistance to etoposide. DSB indicates double-strand break.
Figure Legend Snippet: Schematic representation of the hypoxia-induced mechanisms responsible for cancer cell resistance to etoposide. DSB indicates double-strand break.

Techniques Used:

Effect of hypoxia and/or etoposide on the DNA binding activity of c-myc, NF-κB, and c-jun. HepG2 cells were incubated under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He; 50 µ M) or without etoposide (Nx-Hx) for increasing
Figure Legend Snippet: Effect of hypoxia and/or etoposide on the DNA binding activity of c-myc, NF-κB, and c-jun. HepG2 cells were incubated under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He; 50 µ M) or without etoposide (Nx-Hx) for increasing

Techniques Used: Binding Assay, Activity Assay, Incubation

Effect of p53 silencing on etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM p53 siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx) or hypoxic (Hx) conditions with (Ne-He) or without
Figure Legend Snippet: Effect of p53 silencing on etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM p53 siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx) or hypoxic (Hx) conditions with (Ne-He) or without

Techniques Used: Transfection, Negative Control, Incubation

Effect of p53 silencing on Bak1 expression and effect of Bak1 silencing on the etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM Bak1 siRNA, 50 nM p53 siRNA, or negative control siRNA for 24 hours. Eight hours later, cells were incubated
Figure Legend Snippet: Effect of p53 silencing on Bak1 expression and effect of Bak1 silencing on the etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM Bak1 siRNA, 50 nM p53 siRNA, or negative control siRNA for 24 hours. Eight hours later, cells were incubated

Techniques Used: Expressing, Transfection, Negative Control, Incubation

Effect of c-jun silencing on hypoxia-induced resistance to etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM c-jun siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx; 21% O 2 ) or
Figure Legend Snippet: Effect of c-jun silencing on hypoxia-induced resistance to etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM c-jun siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx; 21% O 2 ) or

Techniques Used: Transfection, Negative Control, Incubation

Effect of hypoxia and/or etoposide on caspase 3 activity and p53 regulation. HepG2 cells were incubated under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He; 50 µ M) or without etoposide (Nx-Hx) for increasing periods from
Figure Legend Snippet: Effect of hypoxia and/or etoposide on caspase 3 activity and p53 regulation. HepG2 cells were incubated under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He; 50 µ M) or without etoposide (Nx-Hx) for increasing periods from

Techniques Used: Activity Assay, Incubation

43) Product Images from "p38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb-Hdm2 interaction and apoptosis"

Article Title: p38 phosphorylates Rb on Ser567 by a novel, cell cycle-independent mechanism that triggers Rb-Hdm2 interaction and apoptosis

Journal: Oncogene

doi: 10.1038/onc.2010.442

p53 is involved in p38 induced apoptosis, but not in p38-mediated phosphorylation and degradation of Rb. ( A ) Mel202s were treated with 12.5 μM of etoposide for 48 h. Scrambled or p53 siRNA was used at a final concentration of 50 nM for 72 h. p53 expression vector or empty vector were transfected for 72 h. Samples were immunoblotted with antibodies against Rb and p53. ( B ) Mel202s were transfected with siRNA against p53 or scrambled siRNA for 72 h and treated with Ad-MKK3 MOI of 15 for 48 h. Samples were immunoblotted with antibodies against Rb and p53. ( C ) Mel202s were transfected with p53siRNA or scrambled siRNA for 72 h. Cells were treated with 12.5 μM of etoposide for 48 h and TUNEL staining was performed. ( D ) Mel202s were transfected with p53 siRNA or scrambled siRNA for 72 h and treated with Ad-LacZ or Ad-MKK3 at an MOI of 15. TUNEL staining was performed.
Figure Legend Snippet: p53 is involved in p38 induced apoptosis, but not in p38-mediated phosphorylation and degradation of Rb. ( A ) Mel202s were treated with 12.5 μM of etoposide for 48 h. Scrambled or p53 siRNA was used at a final concentration of 50 nM for 72 h. p53 expression vector or empty vector were transfected for 72 h. Samples were immunoblotted with antibodies against Rb and p53. ( B ) Mel202s were transfected with siRNA against p53 or scrambled siRNA for 72 h and treated with Ad-MKK3 MOI of 15 for 48 h. Samples were immunoblotted with antibodies against Rb and p53. ( C ) Mel202s were transfected with p53siRNA or scrambled siRNA for 72 h. Cells were treated with 12.5 μM of etoposide for 48 h and TUNEL staining was performed. ( D ) Mel202s were transfected with p53 siRNA or scrambled siRNA for 72 h and treated with Ad-LacZ or Ad-MKK3 at an MOI of 15. TUNEL staining was performed.

Techniques Used: Concentration Assay, Expressing, Plasmid Preparation, Transfection, TUNEL Assay, Staining

Genotoxic stress triggers phosphorylation of Rb on Ser567 by p38. ( A ) Mel202 cells were treated with 12.5 μM of etoposide and 0, 50, 100, or 500 μM of the Cdk2 inhibitor, Roscovitine, for 48 h and then immunoblotted for total Rb and phospho-Rb-567. See Supplemental Figure 1B for confirmation of Roscovitine activity. ( B ) Mel202 cells were treated with p38 inhibitor SB203580 for 48 h and immunoblotted for total Rb and phospho-Rb-567. See Supplemental Figure 1C for confirmation of p38 inhibitor activity. ( C ) siRNA against p38α or p38β was transfected into Mel202 cells at a final concentration of 50 nM for 72 h, all samples were treated with 12.5 μM of etoposide for 48 h, and immunoblotted for Rb. ( D ) Mel202 cells were treated with or without 12.5 μM of etoposide for 48 h and immunoblotted for phospho-p38. ( E ) Mel202 cells were treated with 4 μM etoposide, 30 μM of p38 inhibitor, or both for 48 h. Propidium iodide was used in order to determine the sub-G1 DNA content by flow cytometry. ( F ) Mel202 cells were treated with 12.5 μM etoposide, 30 μM of p38 inhibitor, or both for 48 h. TUNEL staining was performed to determine the percent of apoptotic cells. ( G ) Mel202 cells were treated with Ad-LacZ or Ad-MKK3 at an MOI of 100 with or without 30 μM of p38 inhibitor and immunoblotted for Rb, phospho-Rb-567 or phospho-p38. ( H ) Mel202 cells were treated with Ad-GFP, Ad-cycD1, or Ad-MKK3 at an MOI of 30 for 48 h and immunoblotted for total Rb, phospho-p38, or cyclin D1. ( I ) Flow cytometry was performed using propidium iodide staining to determine the percent of cells in sub-G1. Mel202 cells were treated with Ad-cycD1 or Ad-MKK3 at an MOI of 30 for 48 h. ( J ) Mel501 cells were transfected with SKTT control, Rb, RbΔ780, or RbΔ568 for 72 h and treated with 12.5 μM etoposide for 48 h. TUNEL staining was performed. ( K ) Mel501 cells were transfected with SKTT control, Rb, or RbΔ568 for 72 h and treated with Ad-MKK3 or Ad-LacZ MOI 15 for 48 h. TUNEL staining was performed. Data were normalized to RbΔ568 for each virus.
Figure Legend Snippet: Genotoxic stress triggers phosphorylation of Rb on Ser567 by p38. ( A ) Mel202 cells were treated with 12.5 μM of etoposide and 0, 50, 100, or 500 μM of the Cdk2 inhibitor, Roscovitine, for 48 h and then immunoblotted for total Rb and phospho-Rb-567. See Supplemental Figure 1B for confirmation of Roscovitine activity. ( B ) Mel202 cells were treated with p38 inhibitor SB203580 for 48 h and immunoblotted for total Rb and phospho-Rb-567. See Supplemental Figure 1C for confirmation of p38 inhibitor activity. ( C ) siRNA against p38α or p38β was transfected into Mel202 cells at a final concentration of 50 nM for 72 h, all samples were treated with 12.5 μM of etoposide for 48 h, and immunoblotted for Rb. ( D ) Mel202 cells were treated with or without 12.5 μM of etoposide for 48 h and immunoblotted for phospho-p38. ( E ) Mel202 cells were treated with 4 μM etoposide, 30 μM of p38 inhibitor, or both for 48 h. Propidium iodide was used in order to determine the sub-G1 DNA content by flow cytometry. ( F ) Mel202 cells were treated with 12.5 μM etoposide, 30 μM of p38 inhibitor, or both for 48 h. TUNEL staining was performed to determine the percent of apoptotic cells. ( G ) Mel202 cells were treated with Ad-LacZ or Ad-MKK3 at an MOI of 100 with or without 30 μM of p38 inhibitor and immunoblotted for Rb, phospho-Rb-567 or phospho-p38. ( H ) Mel202 cells were treated with Ad-GFP, Ad-cycD1, or Ad-MKK3 at an MOI of 30 for 48 h and immunoblotted for total Rb, phospho-p38, or cyclin D1. ( I ) Flow cytometry was performed using propidium iodide staining to determine the percent of cells in sub-G1. Mel202 cells were treated with Ad-cycD1 or Ad-MKK3 at an MOI of 30 for 48 h. ( J ) Mel501 cells were transfected with SKTT control, Rb, RbΔ780, or RbΔ568 for 72 h and treated with 12.5 μM etoposide for 48 h. TUNEL staining was performed. ( K ) Mel501 cells were transfected with SKTT control, Rb, or RbΔ568 for 72 h and treated with Ad-MKK3 or Ad-LacZ MOI 15 for 48 h. TUNEL staining was performed. Data were normalized to RbΔ568 for each virus.

Techniques Used: Activity Assay, Transfection, Concentration Assay, Flow Cytometry, Cytometry, TUNEL Assay, Staining

Phosphorylation of Rb by p38 leads to E2F1-mediated apoptosis. ( A ) Mel202 cells were transfected with Rb alone or with MKK3 for 48 h and immunoprecipitation for Rb was performed. The immunoprecipitation was immunoblotted for E2F1. Total lysates were immunoblotted for total Rb and E2F1. ( B ) An E2F1 promoter with three E2F-responsive repeats was used in a luciferase assay. Empty vector (pCDNA3), Rb, E2F1, and constitutively active p38α expression constructs were transfected into Mel202 cells. The star represents constitutively active p38α. ( C ) Scrambled or E2F1 siRNA was used at a final concentration of 50nM for 72 h in Mel501 cells. The cells were treated with 12.5 μM of etoposide for 48 h and TUNEL staining was performed. ( D ) Knock down of E2F1 by siRNA was confirmed in Mel501 cells. 50 nM of scrambled siRNA, 25 nM of E2F1 siRNA, or 50 nM of E2F1 siRNA were transfected for 72 h.
Figure Legend Snippet: Phosphorylation of Rb by p38 leads to E2F1-mediated apoptosis. ( A ) Mel202 cells were transfected with Rb alone or with MKK3 for 48 h and immunoprecipitation for Rb was performed. The immunoprecipitation was immunoblotted for E2F1. Total lysates were immunoblotted for total Rb and E2F1. ( B ) An E2F1 promoter with three E2F-responsive repeats was used in a luciferase assay. Empty vector (pCDNA3), Rb, E2F1, and constitutively active p38α expression constructs were transfected into Mel202 cells. The star represents constitutively active p38α. ( C ) Scrambled or E2F1 siRNA was used at a final concentration of 50nM for 72 h in Mel501 cells. The cells were treated with 12.5 μM of etoposide for 48 h and TUNEL staining was performed. ( D ) Knock down of E2F1 by siRNA was confirmed in Mel501 cells. 50 nM of scrambled siRNA, 25 nM of E2F1 siRNA, or 50 nM of E2F1 siRNA were transfected for 72 h.

Techniques Used: Transfection, Immunoprecipitation, Luciferase, Plasmid Preparation, Expressing, Construct, Concentration Assay, TUNEL Assay, Staining

Phosphorylation of Rb on Ser567 by p38 enhances the Rb-Hdm2 interaction and leads to Hdm2-mediated apoptosis. ( A ) Mel202 cells were treated for 48 h with or without12.5 μM of etoposide. 50 μM of Hdm2 inhibitor was used in lane 2, 5 μM in lane 4, and 10 μM in lane 6. No Hdm2 inhibitor was used in Lanes 1 and 3. ( B ) Mel202 cells were treated with 12.5 μM etoposide for 48 h. Immunoprecipitation for endogenous Hdm2 was performed and samples were immunoblotted for endogenous Rb. Total lysates were immunoblotted for Rb and Hdm2. ( C ) Mel202 cells were treated with Ad-cycD1 or Ad-MKK3 at an MOI of 15 for 48 h, immunoprecipitated for endogenous Hdm2, and immunoblotted for endogenous total Rb. Total lysates were immunoblotted for Rb, phospho-p38, and Hdm2. ( D ) Mel202 cells were transfected with Gal4-tagged Rb constructs. Rb-A (the A domain of Rb, amino acids 379-572, serves as a negative control), Rb, RbΔ567, or RbΔ568. Cells were immunoprecipitated for Gal4 to pull down Rb, and immunoblotted for endogenous Hdm2 and endogenous p53. Total lysates were immunoblotted for Hdm2, p53, and Gal4. ( E ) Mel202 cells were treated with 10 μM Hdm2 inhibitor and 12.5 μM etoposide for 48 h and TUNEL staining was performed. ( F ) Mel202 cells were treated with 10 μM Hdm2 inhibitor for 72 h. Cells were treated Ad-LacZ or Ad-MKK3 at a MOI of 100 for 48 h and TUNEL staining was performed. (G) Mel202 cells were transfected with Hdm2 siRNA for 72 h and treated with 12.5 μM etoposide for 48 h and TUNEL staining was performed. ( H ) Mel202 cells were transfected with Hdm2 siRNA for 72 h and treated with Ad-LacZ or Ad-MKK3 at an MOI of 50 for 48 h and TUNEL staining was performed.
Figure Legend Snippet: Phosphorylation of Rb on Ser567 by p38 enhances the Rb-Hdm2 interaction and leads to Hdm2-mediated apoptosis. ( A ) Mel202 cells were treated for 48 h with or without12.5 μM of etoposide. 50 μM of Hdm2 inhibitor was used in lane 2, 5 μM in lane 4, and 10 μM in lane 6. No Hdm2 inhibitor was used in Lanes 1 and 3. ( B ) Mel202 cells were treated with 12.5 μM etoposide for 48 h. Immunoprecipitation for endogenous Hdm2 was performed and samples were immunoblotted for endogenous Rb. Total lysates were immunoblotted for Rb and Hdm2. ( C ) Mel202 cells were treated with Ad-cycD1 or Ad-MKK3 at an MOI of 15 for 48 h, immunoprecipitated for endogenous Hdm2, and immunoblotted for endogenous total Rb. Total lysates were immunoblotted for Rb, phospho-p38, and Hdm2. ( D ) Mel202 cells were transfected with Gal4-tagged Rb constructs. Rb-A (the A domain of Rb, amino acids 379-572, serves as a negative control), Rb, RbΔ567, or RbΔ568. Cells were immunoprecipitated for Gal4 to pull down Rb, and immunoblotted for endogenous Hdm2 and endogenous p53. Total lysates were immunoblotted for Hdm2, p53, and Gal4. ( E ) Mel202 cells were treated with 10 μM Hdm2 inhibitor and 12.5 μM etoposide for 48 h and TUNEL staining was performed. ( F ) Mel202 cells were treated with 10 μM Hdm2 inhibitor for 72 h. Cells were treated Ad-LacZ or Ad-MKK3 at a MOI of 100 for 48 h and TUNEL staining was performed. (G) Mel202 cells were transfected with Hdm2 siRNA for 72 h and treated with 12.5 μM etoposide for 48 h and TUNEL staining was performed. ( H ) Mel202 cells were transfected with Hdm2 siRNA for 72 h and treated with Ad-LacZ or Ad-MKK3 at an MOI of 50 for 48 h and TUNEL staining was performed.

Techniques Used: Immunoprecipitation, Transfection, Construct, Negative Control, TUNEL Assay, Staining

Loss of Rb leads to apoptosis. ( A ) Mel202 cells were transfected with scrambled siRNA or Rb siRNA, or with vectors expressing cyclin D1 and/or cyclin E to activate endogenous Cdks for 4 days and cell viability assays were performed. ( B ) Cell viability assays were performed in MM28 primary melanoma cells using scrambled siRNA or Rb siRNA. ( C ) Immunoblot analysis confirming effective knock down of Rb with siRNA in Mel202s after 4 days. Tubulin was used as a loading control in all immunoblot experiments. ( D ) 12.5 μM of etoposide treatment halted cell growth in Mel202 cells as shown by growth curves. ( E ) Cleaved caspase 3 staining of Mel202 cells treated with or without 12.5 μM of etoposide for 48 h. ( F ) Flow cytometry was performed using Mel202 cells treated with 0, 1, 5, 12, 25 μM etoposide for 48 h. ( G ) Mel202 cells were treated with 0, 0.6, 6, 12, 25 μM etoposide for 48 h and immunoblotted for cleaved PARP. ( H ) Mel202 cells were treated with 0, 1.5, 3, 6, 12.5, 25, 50 μM etoposide for 48 h and immunoblotted for Rb. ( I ) Mel202 cells were treated with 12.5 μM etoposide for 0, 24, 48, 72 h and immunoblotted for Rb.
Figure Legend Snippet: Loss of Rb leads to apoptosis. ( A ) Mel202 cells were transfected with scrambled siRNA or Rb siRNA, or with vectors expressing cyclin D1 and/or cyclin E to activate endogenous Cdks for 4 days and cell viability assays were performed. ( B ) Cell viability assays were performed in MM28 primary melanoma cells using scrambled siRNA or Rb siRNA. ( C ) Immunoblot analysis confirming effective knock down of Rb with siRNA in Mel202s after 4 days. Tubulin was used as a loading control in all immunoblot experiments. ( D ) 12.5 μM of etoposide treatment halted cell growth in Mel202 cells as shown by growth curves. ( E ) Cleaved caspase 3 staining of Mel202 cells treated with or without 12.5 μM of etoposide for 48 h. ( F ) Flow cytometry was performed using Mel202 cells treated with 0, 1, 5, 12, 25 μM etoposide for 48 h. ( G ) Mel202 cells were treated with 0, 0.6, 6, 12, 25 μM etoposide for 48 h and immunoblotted for cleaved PARP. ( H ) Mel202 cells were treated with 0, 1.5, 3, 6, 12.5, 25, 50 μM etoposide for 48 h and immunoblotted for Rb. ( I ) Mel202 cells were treated with 12.5 μM etoposide for 0, 24, 48, 72 h and immunoblotted for Rb.

Techniques Used: Transfection, Expressing, Staining, Flow Cytometry, Cytometry

44) Product Images from "Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1"

Article Title: Human T-cell leukemia virus type-1-encoded protein HBZ represses p53 function by inhibiting the acetyltransferase activity of p300/CBP and HBO1

Journal: Oncotarget

doi:

Model summarizing the effects of HBZ on p53-regulated transcription of p21/CDKN1A Etoposide-induced DNA damage, stimulates p53-dependent recruitment of p300 and HBO1 to the promoter (dashed blue arrows). During this process p300 acetylates p53 at K382 (ac-labeled arrow) and other lysine residues to increase the DNA-binding activity of p53 (solid blue arrow). HBO1 recruitment may be facilitated by associated ING and JADE/BRPF proteins, which interact with methylated histone H3 through their PHD finger domains. These interactions may position HBO1 in proximity to the transcription start site, where it acetylates histones. By inhibiting the HAT domains of p300 and HBO1, HBZ represses acetylation of p53 and promoter-associated histones. HBZ also restricts the recruitment of HBO1, which may lead to further reduction in acetylation of histones at the promoter. The sum of the effects of HBZ is to dampen the level of activation of p21/CDKN1A (and GADD45A) transcription, leading to a delay in cell cycle arrest induced by etoposide.
Figure Legend Snippet: Model summarizing the effects of HBZ on p53-regulated transcription of p21/CDKN1A Etoposide-induced DNA damage, stimulates p53-dependent recruitment of p300 and HBO1 to the promoter (dashed blue arrows). During this process p300 acetylates p53 at K382 (ac-labeled arrow) and other lysine residues to increase the DNA-binding activity of p53 (solid blue arrow). HBO1 recruitment may be facilitated by associated ING and JADE/BRPF proteins, which interact with methylated histone H3 through their PHD finger domains. These interactions may position HBO1 in proximity to the transcription start site, where it acetylates histones. By inhibiting the HAT domains of p300 and HBO1, HBZ represses acetylation of p53 and promoter-associated histones. HBZ also restricts the recruitment of HBO1, which may lead to further reduction in acetylation of histones at the promoter. The sum of the effects of HBZ is to dampen the level of activation of p21/CDKN1A (and GADD45A) transcription, leading to a delay in cell cycle arrest induced by etoposide.

Techniques Used: Labeling, Binding Assay, Activity Assay, Methylation, HAT Assay, Activation Assay

HBZ delays cell cycle arrest in G2/M following treatment with etoposide A. HBZ does not alter the level of apoptosis induced by etoposide. HCT116 p53 +/+ cells were transiently transfected with an HBZ or empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO) or the DMSO vehicle (control) for 24 hours as indicated. Plots are from a representative experiment and the bar graph shows the average flow cytometry data of early apoptosis (grey bars) and late apoptosis (darker grey bars) from three independent experiments ± S.D. B. HBZ decreases G2/M arrest in an asynchronous cell population. HCT116 p53 +/+ cells were transiently transfected with an HBZ or the empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO), or the DMSO vehicle (control) for 24 hours as indicated. The graph shows the average flow cytometry data from three independent experiments ± S.D. * P
Figure Legend Snippet: HBZ delays cell cycle arrest in G2/M following treatment with etoposide A. HBZ does not alter the level of apoptosis induced by etoposide. HCT116 p53 +/+ cells were transiently transfected with an HBZ or empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO) or the DMSO vehicle (control) for 24 hours as indicated. Plots are from a representative experiment and the bar graph shows the average flow cytometry data of early apoptosis (grey bars) and late apoptosis (darker grey bars) from three independent experiments ± S.D. B. HBZ decreases G2/M arrest in an asynchronous cell population. HCT116 p53 +/+ cells were transiently transfected with an HBZ or the empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO), or the DMSO vehicle (control) for 24 hours as indicated. The graph shows the average flow cytometry data from three independent experiments ± S.D. * P

Techniques Used: Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry

HBZ inhibits acetylation of p53 K382 A. and B. HCT116 p53 +/+ cells were transiently transfected with an HBZ, Tax or an empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO), actinomycin D (ACT.D), doxorubicin (DOX) or the DMSO vehicle control (−) for 8 hours as indicated. Nuclear extracts were prepared and analyzed by Western blot using the antibodies indicated. C. Acetylation of histone H4 by p300 and MOZ. In vitro HAT assays were performed using recombinant histones (2 μM), p300 (2 nM) and MOZ-HAT (0.15 μM) and analyzed by Western blot using antibodies against histone H4 and acetylated histone H4 as indicated. D. Acetylation of p53 by p300 and MOZ. In vitro HAT assays were performed using the same concentrations of recombinant proteins as above, but with p53 (0.1 μM) replacing histones as the substrate. Reactions were analyzed by western blot using antibodies against acetylated lysine, p53 acetyl-K382 and p53. E. HBZ inhibits acetylation of p53 by p300. In vitro HAT assays were performed using recombinant p300 (2 nM), p53 (25 nM) and supplemented with GST (0.3 μM), GST-HBZ (0.3 μM), HBZ-AD (0.3 μM) or HBZ-bZIP (0.3 μM) where indicated. Reactions were analyzed by Western blot using antibodies against acetylated lysine, p53 acetyl-K382 and p53. Identical quantities from the same batch of proteins used in the HAT assay were resolved by SDS-PAGE and stained with Coomassie (lower panel).
Figure Legend Snippet: HBZ inhibits acetylation of p53 K382 A. and B. HCT116 p53 +/+ cells were transiently transfected with an HBZ, Tax or an empty expression vector and, 48 h post-transfection, were treated with etoposide (ETO), actinomycin D (ACT.D), doxorubicin (DOX) or the DMSO vehicle control (−) for 8 hours as indicated. Nuclear extracts were prepared and analyzed by Western blot using the antibodies indicated. C. Acetylation of histone H4 by p300 and MOZ. In vitro HAT assays were performed using recombinant histones (2 μM), p300 (2 nM) and MOZ-HAT (0.15 μM) and analyzed by Western blot using antibodies against histone H4 and acetylated histone H4 as indicated. D. Acetylation of p53 by p300 and MOZ. In vitro HAT assays were performed using the same concentrations of recombinant proteins as above, but with p53 (0.1 μM) replacing histones as the substrate. Reactions were analyzed by western blot using antibodies against acetylated lysine, p53 acetyl-K382 and p53. E. HBZ inhibits acetylation of p53 by p300. In vitro HAT assays were performed using recombinant p300 (2 nM), p53 (25 nM) and supplemented with GST (0.3 μM), GST-HBZ (0.3 μM), HBZ-AD (0.3 μM) or HBZ-bZIP (0.3 μM) where indicated. Reactions were analyzed by Western blot using antibodies against acetylated lysine, p53 acetyl-K382 and p53. Identical quantities from the same batch of proteins used in the HAT assay were resolved by SDS-PAGE and stained with Coomassie (lower panel).

Techniques Used: Transfection, Expressing, Plasmid Preparation, Activated Clotting Time Assay, Western Blot, In Vitro, HAT Assay, Recombinant, SDS Page, Staining

HBO1 contributes to p53-mediated activation of p21/CDKN1A A. HBO1 augments p53-dependent transcription. H1299 cells were cotransfected with pG13-luc (150 ng), and expression vectors for HA-p53 (200 ng), Flag-HBO1 (increasing concentration up to 1 μg), Flag-HBO1 G485 (increasing concentration up to 1 μg), or a combination of those as indicated. The graph shows relative luminescence values ± S.D. from a triplicate experiment. Data are normalized to the control condition artificially set to 1 (lane 1 for the graph on the left and lane 6 for the graph on the right). B. Etoposide induces HBO1 recruitment to the p21/CDKN1A and GADD45A promoters. Chromatin immunoprecipitation (ChIP) analysis was performed on chromatin prepared from untreated HCT116 p53 +/+ cells (grey bars) or cells treated with etoposide (black bars) using p53 and HBO1 antibodies or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis with primers amplifying the BNDF promoter, the p53 binding region in the p21/CDKN1A promoter and the GADD45A promoter. Data are presented as fold enrichment over a control unrelated regions. C. HBO1 recruitment to the p21/CDKN1A and GADD45A promoters is dependent on p53. ChIP analysis in H1299 cells untreated (grey bars) or treated with etoposide (black bars) using an HBO1 antibody or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis with primers amplifying the BNDF promoter, the p53 binding region in the p21/CDKN1A promoter, the GADD45A promoter and an HBO1 binding site in the HOXA9 promoter. D. Characterization of H1299 and HCT116 p53 +/+ cells used in the study by Western blot. E. HBO1 depletion reduces transcription of p21/CDKN1A and GADD45A induced by etoposide. RT-PCR analyses of p21/CDKN1A and GADD45A expression were performed on mRNAs prepared from HCT116 cells transiently transfected with a specific siRNA against HBO1 or a control siRNA and further treated with ETO or not. Expression in each condition is normalized to the condition: -ETO, NT. F. The efficiency of the siRNA was estimated by Western blot.
Figure Legend Snippet: HBO1 contributes to p53-mediated activation of p21/CDKN1A A. HBO1 augments p53-dependent transcription. H1299 cells were cotransfected with pG13-luc (150 ng), and expression vectors for HA-p53 (200 ng), Flag-HBO1 (increasing concentration up to 1 μg), Flag-HBO1 G485 (increasing concentration up to 1 μg), or a combination of those as indicated. The graph shows relative luminescence values ± S.D. from a triplicate experiment. Data are normalized to the control condition artificially set to 1 (lane 1 for the graph on the left and lane 6 for the graph on the right). B. Etoposide induces HBO1 recruitment to the p21/CDKN1A and GADD45A promoters. Chromatin immunoprecipitation (ChIP) analysis was performed on chromatin prepared from untreated HCT116 p53 +/+ cells (grey bars) or cells treated with etoposide (black bars) using p53 and HBO1 antibodies or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis with primers amplifying the BNDF promoter, the p53 binding region in the p21/CDKN1A promoter and the GADD45A promoter. Data are presented as fold enrichment over a control unrelated regions. C. HBO1 recruitment to the p21/CDKN1A and GADD45A promoters is dependent on p53. ChIP analysis in H1299 cells untreated (grey bars) or treated with etoposide (black bars) using an HBO1 antibody or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis with primers amplifying the BNDF promoter, the p53 binding region in the p21/CDKN1A promoter, the GADD45A promoter and an HBO1 binding site in the HOXA9 promoter. D. Characterization of H1299 and HCT116 p53 +/+ cells used in the study by Western blot. E. HBO1 depletion reduces transcription of p21/CDKN1A and GADD45A induced by etoposide. RT-PCR analyses of p21/CDKN1A and GADD45A expression were performed on mRNAs prepared from HCT116 cells transiently transfected with a specific siRNA against HBO1 or a control siRNA and further treated with ETO or not. Expression in each condition is normalized to the condition: -ETO, NT. F. The efficiency of the siRNA was estimated by Western blot.

Techniques Used: Activation Assay, Expressing, Concentration Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection

HBZ inhibits binding of HBO1 to the p21/CDKN1A promoter following etoposide treatment A. Graphic representation of the p21/CDKN1A promoter showing 5′ and 3′ p53 responsive elements (RE, white boxes) and the transcription start site (TSS). Bold horizontal lines denote real-time PCR amplicons. ChIP analyses were performed on chromatin prepared from untreated (grey bars) or etoposide-treated (black bars) cells using an HBO1 antibody or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis. Data are presented as fold enrichment over a control unrelated regions. B. Analysis of a HeLa clonal cell line containing the empty pcDNA 3.1 vector. C. Analysis of a HeLa clonal cell line stably expressing HBZ.
Figure Legend Snippet: HBZ inhibits binding of HBO1 to the p21/CDKN1A promoter following etoposide treatment A. Graphic representation of the p21/CDKN1A promoter showing 5′ and 3′ p53 responsive elements (RE, white boxes) and the transcription start site (TSS). Bold horizontal lines denote real-time PCR amplicons. ChIP analyses were performed on chromatin prepared from untreated (grey bars) or etoposide-treated (black bars) cells using an HBO1 antibody or a preimmune serum (IgG). Precipitated DNA fragments were subjected to real-time PCR analysis. Data are presented as fold enrichment over a control unrelated regions. B. Analysis of a HeLa clonal cell line containing the empty pcDNA 3.1 vector. C. Analysis of a HeLa clonal cell line stably expressing HBZ.

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Plasmid Preparation, Stable Transfection, Expressing

HBZ inhibits p53-mediated activation of p21/CDKN1A and GADD45A HCT116 p53 +/+ cells were transiently transfected with an HBZ or empty expression vector and treated with etoposide (ETO) or the DMSO vehicle control (control) for 8 hours. A. Expression of HAT proteins. Nuclear extracts were analyzed by Western blot using the antibodies indicated. B. mRNA levels of p53-responsive genes. The graph shows real-time PCR data averaged from three or more independent experiments ± S.D. C. mRNA levels of p21/CDKN1A and GADD45A genes. HeLa cells stably transfected with an HBZ or empty expression vector and treated with etoposide (ETO) or the DMSO vehicle control (control) for 6 hours. The graph shows real-time PCR data averaged from four independent experiments ± S.D. * P
Figure Legend Snippet: HBZ inhibits p53-mediated activation of p21/CDKN1A and GADD45A HCT116 p53 +/+ cells were transiently transfected with an HBZ or empty expression vector and treated with etoposide (ETO) or the DMSO vehicle control (control) for 8 hours. A. Expression of HAT proteins. Nuclear extracts were analyzed by Western blot using the antibodies indicated. B. mRNA levels of p53-responsive genes. The graph shows real-time PCR data averaged from three or more independent experiments ± S.D. C. mRNA levels of p21/CDKN1A and GADD45A genes. HeLa cells stably transfected with an HBZ or empty expression vector and treated with etoposide (ETO) or the DMSO vehicle control (control) for 6 hours. The graph shows real-time PCR data averaged from four independent experiments ± S.D. * P

Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, HAT Assay, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection

45) Product Images from "UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4"

Article Title: UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs via downregulation of XRCC4

Journal: Cell Death & Disease

doi: 10.1038/s41419-017-0203-4

XRCC4 downregulation in UHRF1-depleted cells impairs DNA repair and increases drug sensitivity. a DNA ligase IV recruitment onto chromatin upon acute DNA damage. Y79 shCTL and shUHRF1 cells were exposed to high doses of etoposide for 50 min and subjected to immediate cell fractionation. Whole-cell lysates, detergent-soluble (S1) cytosolic fraction, and detergent-insoluble (P2) chromatin fraction were analyzed by immunoblots for the indicated proteins. Tubulin and histones were used as a loading control for cytosolic and chromatin extracts, respectively. b Densitometric analysis for the DNA ligase IV level in cytosolic and chromatin fractions shown in a . The data represent the mean ± SD of fold changes from three independent experiments, relative to the normalized DNA ligase IV level in DMSO-treated shCTL Y79 cells. * P
Figure Legend Snippet: XRCC4 downregulation in UHRF1-depleted cells impairs DNA repair and increases drug sensitivity. a DNA ligase IV recruitment onto chromatin upon acute DNA damage. Y79 shCTL and shUHRF1 cells were exposed to high doses of etoposide for 50 min and subjected to immediate cell fractionation. Whole-cell lysates, detergent-soluble (S1) cytosolic fraction, and detergent-insoluble (P2) chromatin fraction were analyzed by immunoblots for the indicated proteins. Tubulin and histones were used as a loading control for cytosolic and chromatin extracts, respectively. b Densitometric analysis for the DNA ligase IV level in cytosolic and chromatin fractions shown in a . The data represent the mean ± SD of fold changes from three independent experiments, relative to the normalized DNA ligase IV level in DMSO-treated shCTL Y79 cells. * P

Techniques Used: Cell Fractionation, Western Blot

UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs. a – c Dose–response study showing the relative sensitivity to drugs. Stable control-knockdown (shCTL) and UHRF1-knockdown (shUHRF1) Y79 cells were exposed to various concentrations of chemotherapeutic drugs for 48 h, as indicated. d – f Time-course study showing the relative sensitivity to drugs. Cells were treated with 10 µM etoposide ( d ), 0.5 µM camptothecin ( e ), and 100 µM carboplatin ( f ) for the indicated time. The results at each data point are shown as the mean ± standard deviation (SD) of % fold changes from three independent experiments, relative to the cell viability in untreated group. * P
Figure Legend Snippet: UHRF1 depletion sensitizes retinoblastoma cells to chemotherapeutic drugs. a – c Dose–response study showing the relative sensitivity to drugs. Stable control-knockdown (shCTL) and UHRF1-knockdown (shUHRF1) Y79 cells were exposed to various concentrations of chemotherapeutic drugs for 48 h, as indicated. d – f Time-course study showing the relative sensitivity to drugs. Cells were treated with 10 µM etoposide ( d ), 0.5 µM camptothecin ( e ), and 100 µM carboplatin ( f ) for the indicated time. The results at each data point are shown as the mean ± standard deviation (SD) of % fold changes from three independent experiments, relative to the cell viability in untreated group. * P

Techniques Used: Standard Deviation

Enhanced drug sensitivity upon UHRF1 depletion involves apoptotic cell death. a Sub-G1 population detected by flow cytometry in control (shCTL) and UHRF1-knockdown (shUHRF1) Y79 cells treated with vehicle or 10 µM etoposide for 24 h. The percentage of sub-G1 population is shown. b Quantification of early apoptotic cell death by Annexin V-PI staining. Control and UHRF1-knockdown Y79 cells were treated with 10 µM etoposide for the time indicated. The data represent the mean ± SD of % Annexin V + PI − population from triplicate experiments. ** P
Figure Legend Snippet: Enhanced drug sensitivity upon UHRF1 depletion involves apoptotic cell death. a Sub-G1 population detected by flow cytometry in control (shCTL) and UHRF1-knockdown (shUHRF1) Y79 cells treated with vehicle or 10 µM etoposide for 24 h. The percentage of sub-G1 population is shown. b Quantification of early apoptotic cell death by Annexin V-PI staining. Control and UHRF1-knockdown Y79 cells were treated with 10 µM etoposide for the time indicated. The data represent the mean ± SD of % Annexin V + PI − population from triplicate experiments. ** P

Techniques Used: Flow Cytometry, Cytometry, Staining

XRCC4 downregulation in UHRF1-depleted retinoblastoma cells impedes recovery from DNA damage. a Immunoblots for indicated proteins in shCTL and shUHRF1 Y79 cells. Cells were treated with 10 µM etoposide for 24 h. b Expression of XRCC4 in UHRF1-depleted Weri-Rb1, SO-Rb50, and 293T cells after treatment with 10 µM etoposide for 24 h. c qRT-PCR analysis of relative XRCC4 expression in Y79 shCTL and shUHRF1 cells. The bar graph is shown as the mean ± SD of fold changes from five independent experiments, relative to the normalized XRCC4 expression in control-knockdown cell. **** P
Figure Legend Snippet: XRCC4 downregulation in UHRF1-depleted retinoblastoma cells impedes recovery from DNA damage. a Immunoblots for indicated proteins in shCTL and shUHRF1 Y79 cells. Cells were treated with 10 µM etoposide for 24 h. b Expression of XRCC4 in UHRF1-depleted Weri-Rb1, SO-Rb50, and 293T cells after treatment with 10 µM etoposide for 24 h. c qRT-PCR analysis of relative XRCC4 expression in Y79 shCTL and shUHRF1 cells. The bar graph is shown as the mean ± SD of fold changes from five independent experiments, relative to the normalized XRCC4 expression in control-knockdown cell. **** P

Techniques Used: Western Blot, Expressing, Quantitative RT-PCR

UHRF1 depletion does not impair DNA damage sensing. a Immunostaining for γH2AX as an indicator for DNA damage. Y79 shCTL and shUHRF1 cells were pre-treated with either vehicle or 10 µM ATM inhibitor (ATMi) for 2 h, followed by the treatment with 10 µM etoposide for 2 h. b Quantification of γH2AX-positive cells shown in a . Over 300 total cells per group were evaluated for γH2AX-positivity ( > 10 foci/cell), and the data represent the mean ± SD from three independent experiments. ** P
Figure Legend Snippet: UHRF1 depletion does not impair DNA damage sensing. a Immunostaining for γH2AX as an indicator for DNA damage. Y79 shCTL and shUHRF1 cells were pre-treated with either vehicle or 10 µM ATM inhibitor (ATMi) for 2 h, followed by the treatment with 10 µM etoposide for 2 h. b Quantification of γH2AX-positive cells shown in a . Over 300 total cells per group were evaluated for γH2AX-positivity ( > 10 foci/cell), and the data represent the mean ± SD from three independent experiments. ** P

Techniques Used: Immunostaining

46) Product Images from "Role of p53 Serine 46 in p53 Target Gene Regulation"

Article Title: Role of p53 Serine 46 in p53 Target Gene Regulation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017574

Treatment of U2OS cells with Actinomycin D or Etoposide activates p53. (A) Representative cell cycle profiles of U2OS cells untreated or treated for 24 or 48 hours with 5 nM Actinomycin D or 10 µM Etoposide. (B) Western blot showing p53, and p21, cleaved PARP and active Caspase-3 protein levels in whole cell extracts of U2OS cells untreated or treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. TBP is used as loading control. (C) ChIP recovery of p53-binding to the p21 and BAX promoter in U2OS cells treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. ChIP was performed with a p53-antibody (p53-DO1) and qPCR analysis was performed with primers for the respective binding sites. Binding to Myoglobin (myo) was used as a negative control. Error bars represent standard deviation of three individual experiments.
Figure Legend Snippet: Treatment of U2OS cells with Actinomycin D or Etoposide activates p53. (A) Representative cell cycle profiles of U2OS cells untreated or treated for 24 or 48 hours with 5 nM Actinomycin D or 10 µM Etoposide. (B) Western blot showing p53, and p21, cleaved PARP and active Caspase-3 protein levels in whole cell extracts of U2OS cells untreated or treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. TBP is used as loading control. (C) ChIP recovery of p53-binding to the p21 and BAX promoter in U2OS cells treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. ChIP was performed with a p53-antibody (p53-DO1) and qPCR analysis was performed with primers for the respective binding sites. Binding to Myoglobin (myo) was used as a negative control. Error bars represent standard deviation of three individual experiments.

Techniques Used: Western Blot, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Standard Deviation

Global expression analysis upon Actinomycin D and Etoposide treatment. (A) Global expression analysis of U2OS cells treated for 24 hours with Actinomycin D or Etoposide was performed using Affymetrix exon arrays. The ratio of expression changes (log2 signal intensity 24 hours/0 hours) upon Actinomycin D treatment versus Etoposide treatment was plotted against each other. (B) K-Means clustering results of the expression change of genes with a p53-binding site within the transcript or 25 kb up- or downstream upon Actinomycin D or Etoposide treatment. Upregulation is indicated in red; downregulation in green. (C) p53-binding as determined by ChIP-Seq visualized using the UCSC Genome Browser to two genes which are preferentially upregulated upon Etoposide (cluster 4) and two genes which are preferentially upregulated upon Actinomycin D treatment (cluster 5). (D) The number of reads per peak (RPP) of p53-binding upon Actinomycin D is plotted versus the RPP of p53-binding upon Etoposide for the target genes of cluster 4 (left) and cluster 5 (right). To determine the correlation between the RPP of both treatments the square of the sample correlation coefficient between both treatments was calculated (R 2 = 0.69 and R 2 = 0.77 respectively).
Figure Legend Snippet: Global expression analysis upon Actinomycin D and Etoposide treatment. (A) Global expression analysis of U2OS cells treated for 24 hours with Actinomycin D or Etoposide was performed using Affymetrix exon arrays. The ratio of expression changes (log2 signal intensity 24 hours/0 hours) upon Actinomycin D treatment versus Etoposide treatment was plotted against each other. (B) K-Means clustering results of the expression change of genes with a p53-binding site within the transcript or 25 kb up- or downstream upon Actinomycin D or Etoposide treatment. Upregulation is indicated in red; downregulation in green. (C) p53-binding as determined by ChIP-Seq visualized using the UCSC Genome Browser to two genes which are preferentially upregulated upon Etoposide (cluster 4) and two genes which are preferentially upregulated upon Actinomycin D treatment (cluster 5). (D) The number of reads per peak (RPP) of p53-binding upon Actinomycin D is plotted versus the RPP of p53-binding upon Etoposide for the target genes of cluster 4 (left) and cluster 5 (right). To determine the correlation between the RPP of both treatments the square of the sample correlation coefficient between both treatments was calculated (R 2 = 0.69 and R 2 = 0.77 respectively).

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation

Analysis of genome-wide binding profiles of phosphorylated p53. (A–B) Percentage of overall p53-binding sites as obtained from ChIP-seq that are enriched for binding of phosphorylated p53, p53-pS15 or p53-pS46, relative to peak strength. The identified overall p53-binding sites were ranked according to the number of reads per peak and divided into five groups ranking from low to high. For each bin the percentage of p53-binding sites containing also a peak for p53-pS15 (A) or p53-pS46 (B) was plotted. (C–D) Number of reads per peak (RPP) of p53-pS15 (C) and p53-pS46 binding (D) in Actinomycin D versus Etoposide treated cells. To determine the correlation between Actinomycin D versus Etoposide RPP the square of the sample correlation coefficient between both treatments for p53-pS15 (R 2 = 0.82) and p53-pS46 (R 2 = 0.63) was calculated.
Figure Legend Snippet: Analysis of genome-wide binding profiles of phosphorylated p53. (A–B) Percentage of overall p53-binding sites as obtained from ChIP-seq that are enriched for binding of phosphorylated p53, p53-pS15 or p53-pS46, relative to peak strength. The identified overall p53-binding sites were ranked according to the number of reads per peak and divided into five groups ranking from low to high. For each bin the percentage of p53-binding sites containing also a peak for p53-pS15 (A) or p53-pS46 (B) was plotted. (C–D) Number of reads per peak (RPP) of p53-pS15 (C) and p53-pS46 binding (D) in Actinomycin D versus Etoposide treated cells. To determine the correlation between Actinomycin D versus Etoposide RPP the square of the sample correlation coefficient between both treatments for p53-pS15 (R 2 = 0.82) and p53-pS46 (R 2 = 0.63) was calculated.

Techniques Used: Genome Wide, Binding Assay, Chromatin Immunoprecipitation

DNA-binding of p53 phosphorylated at Serine 15 and 46. (A) Western blot showing protein levels of p53, p53 phosphorylated at Serine 15 (p53-pS15) and p53 phosphorylated at Serine 46 (p53-pS46) in whole cell lysates of U2OS cells untreated or treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. TBP is shown as loading control. (B) ChIP-qPCR recovery of p53-pS15 and p53-pS46 binding to the p21 and BAX promoter in U2OS cells treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. ChIP was performed with p53-pS15-antibody or p53-pS46-antibody and qPCR analysis was performed with primers for respective binding sites. Myoglobin (myo) was used as a negative control. Error bars represent standard deviation of three individual experiments. (C) Overlap of p53-pS46 binding sites (left panel) and p53-pS15 binding sites (right panel) of ChIP-seq performed after 5 nM Actinomycin D or 10 µM Etoposide treatment for 24 h using the Genome analyzer (Illumina).
Figure Legend Snippet: DNA-binding of p53 phosphorylated at Serine 15 and 46. (A) Western blot showing protein levels of p53, p53 phosphorylated at Serine 15 (p53-pS15) and p53 phosphorylated at Serine 46 (p53-pS46) in whole cell lysates of U2OS cells untreated or treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. TBP is shown as loading control. (B) ChIP-qPCR recovery of p53-pS15 and p53-pS46 binding to the p21 and BAX promoter in U2OS cells treated for 24 hours with 5 nM Actinomycin D or 10 µM Etoposide. ChIP was performed with p53-pS15-antibody or p53-pS46-antibody and qPCR analysis was performed with primers for respective binding sites. Myoglobin (myo) was used as a negative control. Error bars represent standard deviation of three individual experiments. (C) Overlap of p53-pS46 binding sites (left panel) and p53-pS15 binding sites (right panel) of ChIP-seq performed after 5 nM Actinomycin D or 10 µM Etoposide treatment for 24 h using the Genome analyzer (Illumina).

Techniques Used: Binding Assay, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Standard Deviation

Target genes differentially bound by p53-pS46. (A) ChIP-qPCR recovery of loci which show a higher degree of bound p53 phosphorylated at S46 upon Etoposide treatment in ChIP-seq. U2OS cells were treated with Actinomycin D or Etoposide for 24 hours, before chromatin was isolated. ChIP was performed with the p53-pS46-antibody and qPCR analysis was performed with primers for the putative binding sites. Shown is the recovery of p53-pS46 normalized to the recovery of total p53-DO1 binding in Etoposide or Actinomycin D treated U2OS cells. Error bars represent standard deviation of three individual experiments. (B) Expression change of the respective target genes in U2OS cells treated with Actinomycin D or Etoposide for 24 hours. After cDNA synthesis qPCR was performed and results were normalized against GAPDH expression. Shown is fold over untreated cells. Error bars represent standard deviation of three individual experiments. Asterisks indicate statistical significance shown by Student's t-test (* = P
Figure Legend Snippet: Target genes differentially bound by p53-pS46. (A) ChIP-qPCR recovery of loci which show a higher degree of bound p53 phosphorylated at S46 upon Etoposide treatment in ChIP-seq. U2OS cells were treated with Actinomycin D or Etoposide for 24 hours, before chromatin was isolated. ChIP was performed with the p53-pS46-antibody and qPCR analysis was performed with primers for the putative binding sites. Shown is the recovery of p53-pS46 normalized to the recovery of total p53-DO1 binding in Etoposide or Actinomycin D treated U2OS cells. Error bars represent standard deviation of three individual experiments. (B) Expression change of the respective target genes in U2OS cells treated with Actinomycin D or Etoposide for 24 hours. After cDNA synthesis qPCR was performed and results were normalized against GAPDH expression. Shown is fold over untreated cells. Error bars represent standard deviation of three individual experiments. Asterisks indicate statistical significance shown by Student's t-test (* = P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Binding Assay, Standard Deviation, Expressing

Genome-wide p53-binding profiles. (A) Representative genomic overview of p53-binding to chromosome 19 upon 5 nM Actinomycin D or 10 µM Etoposide treatment for 24 h as determined by ChIP-Seq using the Genome analyzer (Illumina) and visualization using the UCSC genome browser. (B) The number of reads per peak (RPP) of p53-binding upon Actinomycin D is plotted versus the RPP of p53-binding upon Etoposide treatment. To determine the correlation between the RPPs of both treatments the square of the sample correlation coefficient between both treatments was calculated (R 2 = 0.70). (C) The average number of reads per peak (RPP) for the common p53 peaks as well as the treatment preferential peaks upon Actinomycin D or Etoposide treatment are visualized in a boxplot. (D) Genome-wide distribution of the p53-binding sites relative to RefSeq genes. Locations of binding sites are divided in Transcriptional Start Site (TSS) flanking region (5 kb upstream of TSS+first exon+first intron), intragenic region (all introns and exons except first), 5 kb downstream (5 kb downstream of last exon), 5–25 kb up- or downstream of a RefSeq gene or intergenic regions (everything else). The genomic distribution is defined as the number of nucleotides per region divided by the total number of nucleotides in the genome. The asterisk represents significant enrichment compared to overall genomic distribution.
Figure Legend Snippet: Genome-wide p53-binding profiles. (A) Representative genomic overview of p53-binding to chromosome 19 upon 5 nM Actinomycin D or 10 µM Etoposide treatment for 24 h as determined by ChIP-Seq using the Genome analyzer (Illumina) and visualization using the UCSC genome browser. (B) The number of reads per peak (RPP) of p53-binding upon Actinomycin D is plotted versus the RPP of p53-binding upon Etoposide treatment. To determine the correlation between the RPPs of both treatments the square of the sample correlation coefficient between both treatments was calculated (R 2 = 0.70). (C) The average number of reads per peak (RPP) for the common p53 peaks as well as the treatment preferential peaks upon Actinomycin D or Etoposide treatment are visualized in a boxplot. (D) Genome-wide distribution of the p53-binding sites relative to RefSeq genes. Locations of binding sites are divided in Transcriptional Start Site (TSS) flanking region (5 kb upstream of TSS+first exon+first intron), intragenic region (all introns and exons except first), 5 kb downstream (5 kb downstream of last exon), 5–25 kb up- or downstream of a RefSeq gene or intergenic regions (everything else). The genomic distribution is defined as the number of nucleotides per region divided by the total number of nucleotides in the genome. The asterisk represents significant enrichment compared to overall genomic distribution.

Techniques Used: Genome Wide, Binding Assay, Chromatin Immunoprecipitation

47) Product Images from "Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR"

Article Title: Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200604009

PML protein and PML NB dynamics after DSB induction . (A) PML microbody formation occurs rapidly after treatment with etoposide. Two U-2 OS human osteosarcoma cells stably expressing GFP-PML IV protein were imaged by fluorescence microscopy before (T = 0) and after addition of etoposide (20 μM VP16; T = 5 min and T = 2 h). Enlarged region of the cell marked by white asterisk is shown at each time point. White arrowheads indicate newly formed microbodies after VP16 treatment. (B) Formation of PML microbodies in response to DNA DSBs occurs by supramolecular fission from preexisting parental PML NBs. A U-2 OS cell expressing GFP-PML IV was visualized before (T = 0) and during treatment with 20 μM VP16 over several minutes (T = 0.5, 1.0, and 1.5 min are shown). Arrowhead indicates fission of a PML microbody from a larger parental PML NB. (C) PML NBs increase in number in cells irradiated on ice. NHDFs (GM05757s) were incubated on ice for 20 min and either fixed (Control) or irradiated on ice (10 Gy IR) before fixation. Mean PML NB number increases significantly between control (17 ± 1; n = 30) and cells irradiated with 10 Gy IR on ice (24 ± 2; n = 30; *, P = 0.0008). (D) Dynamics of the PML protein within PML NBs is affected by DNA damage and reduced temperature. Asynchronous U-2 OS GFP-PML IV cells were subjected to treatment with etoposide (20 μM VP16 for 30 min) before mobility of PML protein within PML NBs was analyzed by FRAP at 37°C ( n = 20). Mobility of the PML protein at PML NBs in DNA damaged cells is compared with control untreated cells ( n = 20) at 37°C and at 15°C ( n = 7). Data are presented as the mean fluorescence recovery plotted as percent of initial fluorescence intensity of the PML NB over 14 min. Error bars represent the standard error. Bars, 5 μm.
Figure Legend Snippet: PML protein and PML NB dynamics after DSB induction . (A) PML microbody formation occurs rapidly after treatment with etoposide. Two U-2 OS human osteosarcoma cells stably expressing GFP-PML IV protein were imaged by fluorescence microscopy before (T = 0) and after addition of etoposide (20 μM VP16; T = 5 min and T = 2 h). Enlarged region of the cell marked by white asterisk is shown at each time point. White arrowheads indicate newly formed microbodies after VP16 treatment. (B) Formation of PML microbodies in response to DNA DSBs occurs by supramolecular fission from preexisting parental PML NBs. A U-2 OS cell expressing GFP-PML IV was visualized before (T = 0) and during treatment with 20 μM VP16 over several minutes (T = 0.5, 1.0, and 1.5 min are shown). Arrowhead indicates fission of a PML microbody from a larger parental PML NB. (C) PML NBs increase in number in cells irradiated on ice. NHDFs (GM05757s) were incubated on ice for 20 min and either fixed (Control) or irradiated on ice (10 Gy IR) before fixation. Mean PML NB number increases significantly between control (17 ± 1; n = 30) and cells irradiated with 10 Gy IR on ice (24 ± 2; n = 30; *, P = 0.0008). (D) Dynamics of the PML protein within PML NBs is affected by DNA damage and reduced temperature. Asynchronous U-2 OS GFP-PML IV cells were subjected to treatment with etoposide (20 μM VP16 for 30 min) before mobility of PML protein within PML NBs was analyzed by FRAP at 37°C ( n = 20). Mobility of the PML protein at PML NBs in DNA damaged cells is compared with control untreated cells ( n = 20) at 37°C and at 15°C ( n = 7). Data are presented as the mean fluorescence recovery plotted as percent of initial fluorescence intensity of the PML NB over 14 min. Error bars represent the standard error. Bars, 5 μm.

Techniques Used: Stable Transfection, Expressing, Fluorescence, Microscopy, Irradiation, Incubation

PML NB induction in response to DSBs requires NBS1, Chk2, and ATR-function . Cells were treated with etoposide (20 μM VP16 for 30 min), left to recover for 3 h, and processed for IF detection of PML. DNA was counterstained with DAPI. PML NB number is indicated in maximum-intensity Z projections of IF images of control and etoposide-treated cells (left) and a comparison of mean PML NB number (right) is shown. Error bars represent the SEM. Bars, 5 μm. (A) Comparison of the PML NB number between etoposide-treated Chk2-null (Chk2 −/−) and wild-type Chk2 (Chk2 WT) MEFs (*, P
Figure Legend Snippet: PML NB induction in response to DSBs requires NBS1, Chk2, and ATR-function . Cells were treated with etoposide (20 μM VP16 for 30 min), left to recover for 3 h, and processed for IF detection of PML. DNA was counterstained with DAPI. PML NB number is indicated in maximum-intensity Z projections of IF images of control and etoposide-treated cells (left) and a comparison of mean PML NB number (right) is shown. Error bars represent the SEM. Bars, 5 μm. (A) Comparison of the PML NB number between etoposide-treated Chk2-null (Chk2 −/−) and wild-type Chk2 (Chk2 WT) MEFs (*, P

Techniques Used:

The increase in PML NB number in response to DSBs is independent of new protein translation and p53 . NHDF cells (GM05757) in the presence or absence of 150 μM cycloheximide (CHX), Saos-2 human osteosarcoma cells, and isogenic HCT116 human colon carcinoma cells (+ or − p53) were treated with etoposide (20 μM VP16) for 30 min (*, P
Figure Legend Snippet: The increase in PML NB number in response to DSBs is independent of new protein translation and p53 . NHDF cells (GM05757) in the presence or absence of 150 μM cycloheximide (CHX), Saos-2 human osteosarcoma cells, and isogenic HCT116 human colon carcinoma cells (+ or − p53) were treated with etoposide (20 μM VP16) for 30 min (*, P

Techniques Used:

Ultrastructural analysis of PML NBs in NHDFs by correlative LM/ESI before and after etoposide-induced DNA damage . Regions of interest containing a PML NB, which are shown at higher magnification in subsequent images, are delineated by white boxes. (A) LM/ESI of a single NHDF (GM05757) cell, fluorescently labeled for PML protein. Elemental maps of nitrogen (N) and phosphorus (P), and the merged maps of a PML NB and its surrounding nucleoplasm reveal protein-based (cyan) and nucleic acid-based (yellow) components. Chromatin appears yellow in the merged image because of high N and P content. A single PML NB is shown at higher magnification (cyan, as indicated by the arrow) making many contacts to the surrounding chromatin (yellow), and has radial symmetry typical of PML NBs in unstressed cells. (B) LM/ESI of a single NHDF (GM05757) treated with 20 μM etoposide (VP16) for 30 min, fluorescently labeled for PML protein. After treatment with VP16, the protein core of PML NB is disrupted in response to DSB induction; few contacts with chromatin remain, and radial symmetry is lost. (C) PML NB in B, at higher magnification (left), and a cartoon representation of the same EM micrograph (right), where PML protein–containing protein structures (red), chromatin (yellow), and other nonchromosomal protein (blue) are shown. Redistribution of PML microbodies along chromatin fibers (asterisks) is observed, and larger interchromatin spaces (black areas) are apparent. PML protein localization was determined by immunogold detection of PML (white dots). Bars, 500 nm.
Figure Legend Snippet: Ultrastructural analysis of PML NBs in NHDFs by correlative LM/ESI before and after etoposide-induced DNA damage . Regions of interest containing a PML NB, which are shown at higher magnification in subsequent images, are delineated by white boxes. (A) LM/ESI of a single NHDF (GM05757) cell, fluorescently labeled for PML protein. Elemental maps of nitrogen (N) and phosphorus (P), and the merged maps of a PML NB and its surrounding nucleoplasm reveal protein-based (cyan) and nucleic acid-based (yellow) components. Chromatin appears yellow in the merged image because of high N and P content. A single PML NB is shown at higher magnification (cyan, as indicated by the arrow) making many contacts to the surrounding chromatin (yellow), and has radial symmetry typical of PML NBs in unstressed cells. (B) LM/ESI of a single NHDF (GM05757) treated with 20 μM etoposide (VP16) for 30 min, fluorescently labeled for PML protein. After treatment with VP16, the protein core of PML NB is disrupted in response to DSB induction; few contacts with chromatin remain, and radial symmetry is lost. (C) PML NB in B, at higher magnification (left), and a cartoon representation of the same EM micrograph (right), where PML protein–containing protein structures (red), chromatin (yellow), and other nonchromosomal protein (blue) are shown. Redistribution of PML microbodies along chromatin fibers (asterisks) is observed, and larger interchromatin spaces (black areas) are apparent. PML protein localization was determined by immunogold detection of PML (white dots). Bars, 500 nm.

Techniques Used: Labeling

PML NB number increases in response to DSBs in NHDFs . NHDF cells (GM05757) were treated with varying doses of IR, etoposide (20 μM VP16), or 1.5 μM doxorubicin for 30 min to induce DSBs. (A) IF analysis of PML NB number in maximum-intensity Z projections of NHDFs after etoposide; time after treatment is indicated in hours. (B) IF analysis of the distribution of PML NBs in relation to DSBs in NHDFs after 2 Gy IR or VP16. γ-H2AX is used as a marker for chromatin containing DSBs, and asterisks mark the time points in which the maximum fluorescence intensity of γ-H2AX was first detected. Arrowheads indicate juxtaposition of γ-H2AX and PML NBs at 18 h after DNA damage (inset). Images represent a single focal plane. (C) Comparison of mean PML NB number over time after IR, VP16, and doxorubicin treatment. (D) Comparison of fold increase in PML NB number over time after IR, VP16, and doxorubicin treatment. (E and F) Response of PML NBs to graded doses of IR expressed as a function of time (E) or at each time point as a function of dose (F). Bars, 5 μm.
Figure Legend Snippet: PML NB number increases in response to DSBs in NHDFs . NHDF cells (GM05757) were treated with varying doses of IR, etoposide (20 μM VP16), or 1.5 μM doxorubicin for 30 min to induce DSBs. (A) IF analysis of PML NB number in maximum-intensity Z projections of NHDFs after etoposide; time after treatment is indicated in hours. (B) IF analysis of the distribution of PML NBs in relation to DSBs in NHDFs after 2 Gy IR or VP16. γ-H2AX is used as a marker for chromatin containing DSBs, and asterisks mark the time points in which the maximum fluorescence intensity of γ-H2AX was first detected. Arrowheads indicate juxtaposition of γ-H2AX and PML NBs at 18 h after DNA damage (inset). Images represent a single focal plane. (C) Comparison of mean PML NB number over time after IR, VP16, and doxorubicin treatment. (D) Comparison of fold increase in PML NB number over time after IR, VP16, and doxorubicin treatment. (E and F) Response of PML NBs to graded doses of IR expressed as a function of time (E) or at each time point as a function of dose (F). Bars, 5 μm.

Techniques Used: Marker, Fluorescence

The increase in PML NB number in response to DSBs is delayed or inhibited in the presence of PI3 kinase inhibitors and in DNA repair–deficient cell lines . (A) Comparison of effects of DNA repair kinase inhibitors on the increase in PML NB number in response to DSBs. NHDF cell line GM05757 (control) was pretreated with 10 μM Chk2 kinase inhibitor (Chk2 inhibitor II) or various PI3 kinase inhibitors (5 mM caffeine, 20 μM wortmannin, or 50 μM LY2942002) for 30 min before treatment with etoposide (20 μM VP16 for 30 min; *, P
Figure Legend Snippet: The increase in PML NB number in response to DSBs is delayed or inhibited in the presence of PI3 kinase inhibitors and in DNA repair–deficient cell lines . (A) Comparison of effects of DNA repair kinase inhibitors on the increase in PML NB number in response to DSBs. NHDF cell line GM05757 (control) was pretreated with 10 μM Chk2 kinase inhibitor (Chk2 inhibitor II) or various PI3 kinase inhibitors (5 mM caffeine, 20 μM wortmannin, or 50 μM LY2942002) for 30 min before treatment with etoposide (20 μM VP16 for 30 min; *, P

Techniques Used:

48) Product Images from "Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death"

Article Title: Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death

Journal: Scientific Reports

doi: 10.1038/srep15623

Doxorubicin (DOX) and etoposide (ETO) induced nucleus swelling, while differently affecting nucleus morphology. ( A ) Schematic of nucleus swelling and nuclear envelope topography measured using a hemocytometer, confocal microscopy, and CNT/AFM probes systems. ( B ) DOX- and ETO-treated cells were harvested and the nuclei were extracted. Nuclear extracts were seeded on a hemocytometer and visually measured using phase contrast microscopy (scale bar represents 50 μm). ( C ) Nuclear extracts were seeded on coverslips for 15 min and stained with nucleus targeting dies such as Hoechst 33258 and PI dyes that were detected using confocal microscopy. Representative images shown are the merged images for staining with Hoechst 33258 (blue color) and PI (red color; scale bar represents 10 μm).
Figure Legend Snippet: Doxorubicin (DOX) and etoposide (ETO) induced nucleus swelling, while differently affecting nucleus morphology. ( A ) Schematic of nucleus swelling and nuclear envelope topography measured using a hemocytometer, confocal microscopy, and CNT/AFM probes systems. ( B ) DOX- and ETO-treated cells were harvested and the nuclei were extracted. Nuclear extracts were seeded on a hemocytometer and visually measured using phase contrast microscopy (scale bar represents 50 μm). ( C ) Nuclear extracts were seeded on coverslips for 15 min and stained with nucleus targeting dies such as Hoechst 33258 and PI dyes that were detected using confocal microscopy. Representative images shown are the merged images for staining with Hoechst 33258 (blue color) and PI (red color; scale bar represents 10 μm).

Techniques Used: Confocal Microscopy, Microscopy, Staining

Doxorubicin (DOX) and etoposide (ETO) induced cell swelling and necrotic plasma membrane ruptures. ( A ) DOX- and ETO-treated cells were seeded in culture dishes for 3 h, and cell swelling was measured by phase contrast microscopy (scale bar represents 100 μm). ( B ) DOX- and ETO-treated cells were harvested and seeded on culture dishes for 3 h, after which topography changes in the plasma membrane were measured using CNT/AFM probes. Images represent the 3D topography (45- and 10-μm scale) and enhanced color topography (45-μm scale) using the XEI software, which is described in Supplementary Fig. S3 .
Figure Legend Snippet: Doxorubicin (DOX) and etoposide (ETO) induced cell swelling and necrotic plasma membrane ruptures. ( A ) DOX- and ETO-treated cells were seeded in culture dishes for 3 h, and cell swelling was measured by phase contrast microscopy (scale bar represents 100 μm). ( B ) DOX- and ETO-treated cells were harvested and seeded on culture dishes for 3 h, after which topography changes in the plasma membrane were measured using CNT/AFM probes. Images represent the 3D topography (45- and 10-μm scale) and enhanced color topography (45-μm scale) using the XEI software, which is described in Supplementary Fig. S3 .

Techniques Used: Microscopy, Software

Inhibition of pan-caspase regained etoposide (ETO)-induced loss of cell adhesion. ( A ) Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay and detected with a luminescence reader in ETO-treated cells co-treated with z-VAD, and the intensity is shown in the image. ( B ) Cells treated with ETO (green color) and co-treated with z-VAD (caspase inhibitor, blue color) were harvested at 72 h and cell adhesion, including cell index (CI) and saturation times (ST), were measured after detection by the xCELLigence system for 10 s intervals and monitored for 205 min. Black closed diagram indicate saturation (±1%) times. ( C , D ) The table shows cell size (FSC), adhesion area (CI), and saturation times (ST; h) at which cell adhesion was examined using computational analysis when compared to the control. The histogram shows cell adhesion levels using the derived formula for calculations (statistical analysis P-value of *P
Figure Legend Snippet: Inhibition of pan-caspase regained etoposide (ETO)-induced loss of cell adhesion. ( A ) Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay and detected with a luminescence reader in ETO-treated cells co-treated with z-VAD, and the intensity is shown in the image. ( B ) Cells treated with ETO (green color) and co-treated with z-VAD (caspase inhibitor, blue color) were harvested at 72 h and cell adhesion, including cell index (CI) and saturation times (ST), were measured after detection by the xCELLigence system for 10 s intervals and monitored for 205 min. Black closed diagram indicate saturation (±1%) times. ( C , D ) The table shows cell size (FSC), adhesion area (CI), and saturation times (ST; h) at which cell adhesion was examined using computational analysis when compared to the control. The histogram shows cell adhesion levels using the derived formula for calculations (statistical analysis P-value of *P

Techniques Used: Inhibition, Activity Assay, Caspase-Glo Assay, Derivative Assay

Doxorubicin (DOX) and etoposide (ETO) induced nucleus swelling and changes in area and volume, while ETO triggered nuclear envelope rupturing and DNA leakage. ( A ) Nuclear extracts were seeded on culture dishes for 15 min, and the nuclear envelope topography of fixed nuclei was measured using a CNT/AFM probe system. Images shown represent the 3D topography (30- and 15-μm scale), enhanced color topography (10-μm scale), and line profiles (10-μm scale). See also Supplementary Fig. S5 . ( B ) Images show the DNA released in nuclei with 3D topography (2- to 10-μm scales), 2D topography (1-μm scale), and line profiles. ( C , D ) Table and histograms show the average nucleus area (μm 2 ) and nucleus volume (μm 3 ) analyzed using XEI software in 10 nuclei (statistical analysis P-value of *P
Figure Legend Snippet: Doxorubicin (DOX) and etoposide (ETO) induced nucleus swelling and changes in area and volume, while ETO triggered nuclear envelope rupturing and DNA leakage. ( A ) Nuclear extracts were seeded on culture dishes for 15 min, and the nuclear envelope topography of fixed nuclei was measured using a CNT/AFM probe system. Images shown represent the 3D topography (30- and 15-μm scale), enhanced color topography (10-μm scale), and line profiles (10-μm scale). See also Supplementary Fig. S5 . ( B ) Images show the DNA released in nuclei with 3D topography (2- to 10-μm scales), 2D topography (1-μm scale), and line profiles. ( C , D ) Table and histograms show the average nucleus area (μm 2 ) and nucleus volume (μm 3 ) analyzed using XEI software in 10 nuclei (statistical analysis P-value of *P

Techniques Used: Software

Inhibition of pan-caspase suppressed nuclear envelope rupturing and destruction of the nuclear pore complex, and regained endonuclease G translocation in the nuclei by etoposide (ETO). ( A ) Cells treated with ETO and cells co-treated with z-VAD were harvested. Nuclear extracts were seeded on culture dishes for 15 min and the nuclei were fixed. We measured nuclear envelope topography using a CNT/AFM probes system. Images shown are representative of 3D topography at 30-, 10-, and 2-μm scales. See also Supplementary Fig. S7 . ( B ) Cells treated with ETO and co-treated with z-VAD regulated endonuclease G (green color) translocation levels, indicated by immunofluorescence staining and Hoechst 33258 (blue color) that was used for nuclear staining and were measured using confocal microscopy (scale bar represents 10 μm) at 72 h. Arrow indicates nuclear envelope ruptures (collapse Hoechst 33258 intensity) and/or endonuclease G translocation which also is also shown in Supplementary Fig. S8 . ( C ) Translocation endonuclease G levels stained by immunofluorescence and Hoechst 33258 for nuclei that were measured using the Cellomics ArrayScan HCS Reader for at least 200 cells. Endonuclease G intensity was dependent on Hoechst 33258-positive area that is shown in the histogram compared to the control (statistical analysis P-value of *P
Figure Legend Snippet: Inhibition of pan-caspase suppressed nuclear envelope rupturing and destruction of the nuclear pore complex, and regained endonuclease G translocation in the nuclei by etoposide (ETO). ( A ) Cells treated with ETO and cells co-treated with z-VAD were harvested. Nuclear extracts were seeded on culture dishes for 15 min and the nuclei were fixed. We measured nuclear envelope topography using a CNT/AFM probes system. Images shown are representative of 3D topography at 30-, 10-, and 2-μm scales. See also Supplementary Fig. S7 . ( B ) Cells treated with ETO and co-treated with z-VAD regulated endonuclease G (green color) translocation levels, indicated by immunofluorescence staining and Hoechst 33258 (blue color) that was used for nuclear staining and were measured using confocal microscopy (scale bar represents 10 μm) at 72 h. Arrow indicates nuclear envelope ruptures (collapse Hoechst 33258 intensity) and/or endonuclease G translocation which also is also shown in Supplementary Fig. S8 . ( C ) Translocation endonuclease G levels stained by immunofluorescence and Hoechst 33258 for nuclei that were measured using the Cellomics ArrayScan HCS Reader for at least 200 cells. Endonuclease G intensity was dependent on Hoechst 33258-positive area that is shown in the histogram compared to the control (statistical analysis P-value of *P

Techniques Used: Inhibition, Translocation Assay, Immunofluorescence, Staining, Confocal Microscopy

Doxorubicin (DOX) and etoposide (ETO) induced cell swelling, while differently regulating cell adhesion. ( A ) Schematic of necrosis and nepoptosis regulation of cell swelling, cell adhesion, and morphological changes measured using a hemocytometer, FACS, the xCELLigence system, and CNT/AFM probes. ( B ) DOX- and ETO-induced cell swelling were visually measured using hemocytometer analysis and detected by phase contrast microscopy (scale bar represents 250 μm) at 72 h. ( C ) DOX- and ETO-induced numerical cell size measured using FSC units of analysis for increasing times. ( D ) DOX- (red color) and ETO (green color)-treated cells were harvested after 72 h and cell adhesion was measured, including cell index (CI) and saturation times (ST) detected using xCELLigence for 10 s intervals and monitored for 3 h. Black closed diagram indicate saturation (±1.0%) times. ( E , F ) Table shows the cell size (FSC), adhesion area (CI), and saturation times (ST; h) at which cell adhesion was examined using computational analysis (described in results) compared to the control. The histogram shows cell adhesion levels calculated using the derived formula (described in results) for DOX- and ETO-treated cells for 72 h. ( G ) DOX-and ETO-treated cells were seeded in culture dishes for 3 h. Expression level of F-actin (red) was measured using immunofluorescence staining and detected by confocal microscopy. Hoechst 33258 (blue) was used for nuclear staining and scale bar represents 50 μm. All histograms represent statistical analysis (P-value of *P
Figure Legend Snippet: Doxorubicin (DOX) and etoposide (ETO) induced cell swelling, while differently regulating cell adhesion. ( A ) Schematic of necrosis and nepoptosis regulation of cell swelling, cell adhesion, and morphological changes measured using a hemocytometer, FACS, the xCELLigence system, and CNT/AFM probes. ( B ) DOX- and ETO-induced cell swelling were visually measured using hemocytometer analysis and detected by phase contrast microscopy (scale bar represents 250 μm) at 72 h. ( C ) DOX- and ETO-induced numerical cell size measured using FSC units of analysis for increasing times. ( D ) DOX- (red color) and ETO (green color)-treated cells were harvested after 72 h and cell adhesion was measured, including cell index (CI) and saturation times (ST) detected using xCELLigence for 10 s intervals and monitored for 3 h. Black closed diagram indicate saturation (±1.0%) times. ( E , F ) Table shows the cell size (FSC), adhesion area (CI), and saturation times (ST; h) at which cell adhesion was examined using computational analysis (described in results) compared to the control. The histogram shows cell adhesion levels calculated using the derived formula (described in results) for DOX- and ETO-treated cells for 72 h. ( G ) DOX-and ETO-treated cells were seeded in culture dishes for 3 h. Expression level of F-actin (red) was measured using immunofluorescence staining and detected by confocal microscopy. Hoechst 33258 (blue) was used for nuclear staining and scale bar represents 50 μm. All histograms represent statistical analysis (P-value of *P

Techniques Used: FACS, Microscopy, Derivative Assay, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Significance Assay

49) Product Images from "CD26/dipeptidyl peptidase IV enhances expression of topoisomerase II alpha and sensitivity to apoptosis induced by topoisomerase II inhibitors"

Article Title: CD26/dipeptidyl peptidase IV enhances expression of topoisomerase II alpha and sensitivity to apoptosis induced by topoisomerase II inhibitors

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601253

Effect of caspase-9 inhibitor z-LEHD-fmk on etoposide-induced apoptosis in wtCD26 Jurkat transfectant. wtCD26 Jurkat transfectants were incubated at 37°C for 2 h of preincubation with z-LEHD-fmk at varying doses, and then treated with 3 μ M etoposide or 1 μ M doxorubicin for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP, caspase-3, caspase-9, and β -actin were performed as described in Materials and Methods ( * ): caspase-3 cleaved products. Each lane was loaded with 30 μ g of protein.
Figure Legend Snippet: Effect of caspase-9 inhibitor z-LEHD-fmk on etoposide-induced apoptosis in wtCD26 Jurkat transfectant. wtCD26 Jurkat transfectants were incubated at 37°C for 2 h of preincubation with z-LEHD-fmk at varying doses, and then treated with 3 μ M etoposide or 1 μ M doxorubicin for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP, caspase-3, caspase-9, and β -actin were performed as described in Materials and Methods ( * ): caspase-3 cleaved products. Each lane was loaded with 30 μ g of protein.

Techniques Used: Transfection, Incubation, SDS Page

CD26/DPPIV-associated enhancement in PARP cleavage induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C with media containing etoposide for 16 h or doxorubicin for 18 h at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained. Following SDS–PAGE of lysates, immunoblotting studies for PARP and β -actin were performed as described in Materials and Methods. The cleaved product of PARP was detected at ∼85 kDa. Each lane was loaded with 30 μ g of protein.
Figure Legend Snippet: CD26/DPPIV-associated enhancement in PARP cleavage induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C with media containing etoposide for 16 h or doxorubicin for 18 h at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained. Following SDS–PAGE of lysates, immunoblotting studies for PARP and β -actin were performed as described in Materials and Methods. The cleaved product of PARP was detected at ∼85 kDa. Each lane was loaded with 30 μ g of protein.

Techniques Used: Incubation, SDS Page

Enhancing effect of CD26/DPPIV surface expression on apoptosis induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C in culture media alone or culture media containing etoposide ( A ) for 14 h or doxorubicin ( B ) for 16 h at the concentrations indicated. Cells were then harvested and Annexin V/PI assays were performed as described in Materials and Methods. wtCD26 : wild-type CD26 Jurkat transfectant; S630A : Jurkat cells transfected with mutant CD26 containing an alanine at the putative catalytic serine residue at position 630, resulting in a mutant CD26-positive/DPPIV-negative Jurkat transfectant; control : nontransfected parental Jurkat; 340–4 : Jurkat cells transfected with mutant CD26 containing point mutations at the ADA-binding site residues 340–343, with amino acids L 340 , V 341 , A 342 , and R 343 being replaced by amino acids P 340 , S 341 , E 342 , and Q 343 , resulting in a mutant CD26-positive/DPPIV-positive mutant CD26 Jurkat transfectant incapable of binding ADA. Data are representative of three separate experiments. ( C ) wtCD26 Jurkat transfectants and parental cells were treated with doxorubicin over the indicated time intervals and drug concentrations. a: 12 h, b: 24 h, c: 36 h. Data are representative of three separate experiments.
Figure Legend Snippet: Enhancing effect of CD26/DPPIV surface expression on apoptosis induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C in culture media alone or culture media containing etoposide ( A ) for 14 h or doxorubicin ( B ) for 16 h at the concentrations indicated. Cells were then harvested and Annexin V/PI assays were performed as described in Materials and Methods. wtCD26 : wild-type CD26 Jurkat transfectant; S630A : Jurkat cells transfected with mutant CD26 containing an alanine at the putative catalytic serine residue at position 630, resulting in a mutant CD26-positive/DPPIV-negative Jurkat transfectant; control : nontransfected parental Jurkat; 340–4 : Jurkat cells transfected with mutant CD26 containing point mutations at the ADA-binding site residues 340–343, with amino acids L 340 , V 341 , A 342 , and R 343 being replaced by amino acids P 340 , S 341 , E 342 , and Q 343 , resulting in a mutant CD26-positive/DPPIV-positive mutant CD26 Jurkat transfectant incapable of binding ADA. Data are representative of three separate experiments. ( C ) wtCD26 Jurkat transfectants and parental cells were treated with doxorubicin over the indicated time intervals and drug concentrations. a: 12 h, b: 24 h, c: 36 h. Data are representative of three separate experiments.

Techniques Used: Expressing, Incubation, Transfection, Mutagenesis, Binding Assay

Effect of CD26/DPPIV on DR5 expression induced by etoposide treatment. ( A ) Jurkat cells were incubated at 37°C in culture media containing etoposide (3 μ M ) for the indicated time periods at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5 and β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein. Anti- DR5 mAb detects two bands of 58 and 32 kDa. ( B ) Following 2 h of preincubation at 37°C with varying doses of z-LEHD-fmk, wtCD26 Jurkat transfectants were treated with 3 μ M etoposide or 1 μ M doxorubicin for 48 h. Cells were then harvested, and whole cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5, caspase-9, and β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein.
Figure Legend Snippet: Effect of CD26/DPPIV on DR5 expression induced by etoposide treatment. ( A ) Jurkat cells were incubated at 37°C in culture media containing etoposide (3 μ M ) for the indicated time periods at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5 and β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein. Anti- DR5 mAb detects two bands of 58 and 32 kDa. ( B ) Following 2 h of preincubation at 37°C with varying doses of z-LEHD-fmk, wtCD26 Jurkat transfectants were treated with 3 μ M etoposide or 1 μ M doxorubicin for 48 h. Cells were then harvested, and whole cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5, caspase-9, and β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein.

Techniques Used: Expressing, Incubation, SDS Page

CD26-associated enhancement of doxorubicin or etoposide-induced PARP cleavage. Parental Jurkat cells were incubated overnight in culture media alone (−) or culture media containing soluble CD26 (sCD26) molecules (300 μ g ml −1 ) (+) at 37°C, followed by incubation with doxorubicin or etoposide at the indicated concentrations for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP or β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein.
Figure Legend Snippet: CD26-associated enhancement of doxorubicin or etoposide-induced PARP cleavage. Parental Jurkat cells were incubated overnight in culture media alone (−) or culture media containing soluble CD26 (sCD26) molecules (300 μ g ml −1 ) (+) at 37°C, followed by incubation with doxorubicin or etoposide at the indicated concentrations for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP or β -actin were performed as described in Materials and Methods. Each lane was loaded with 30 μ g of protein.

Techniques Used: Incubation, SDS Page

Time course study of the effect of CD26/DPPIV surface expression on etoposide-induced apoptosis. Jurkat cells were incubated at 37°C with media containing 3 μ M etoposide or 1 μ M doxorubicin for the indicated time periods at the indicated doses. Cells were then harvested, and cytosol fractions were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies with specific antibodies for PARP, caspase-9, caspase-3, Apaf-1, Bcl-xl, and β -actin were performed as described in Materials and methods ( * ): caspase-3 cleaved products; ( ** ): Bcl-xl cleaved products. Each lane was loaded with 30 μ g of protein.
Figure Legend Snippet: Time course study of the effect of CD26/DPPIV surface expression on etoposide-induced apoptosis. Jurkat cells were incubated at 37°C with media containing 3 μ M etoposide or 1 μ M doxorubicin for the indicated time periods at the indicated doses. Cells were then harvested, and cytosol fractions were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies with specific antibodies for PARP, caspase-9, caspase-3, Apaf-1, Bcl-xl, and β -actin were performed as described in Materials and methods ( * ): caspase-3 cleaved products; ( ** ): Bcl-xl cleaved products. Each lane was loaded with 30 μ g of protein.

Techniques Used: Expressing, Incubation, SDS Page

50) Product Images from "Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents"

Article Title: Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1013754107

Tubacin up-regulates DDIT3 and DDIT4 , down-regulates replication proteins, and induces a G1 arrest. ( A ) Real-time PCR analysis on LNCaP cells cultured with tubacin, SAHA, etoposide, and the combinations of tubacin with SAHA or etoposide for 24 h. Primers
Figure Legend Snippet: Tubacin up-regulates DDIT3 and DDIT4 , down-regulates replication proteins, and induces a G1 arrest. ( A ) Real-time PCR analysis on LNCaP cells cultured with tubacin, SAHA, etoposide, and the combinations of tubacin with SAHA or etoposide for 24 h. Primers

Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture

Tubacin enhances the accumulation of γH2AX and phospho-Chk2 induced by SAHA or etoposide. ( A ) Western blot analysis showing accumulation of γH2AX following a 24-h culture with tubacin, SAHA, etoposide, and the combinations of tubacin with
Figure Legend Snippet: Tubacin enhances the accumulation of γH2AX and phospho-Chk2 induced by SAHA or etoposide. ( A ) Western blot analysis showing accumulation of γH2AX following a 24-h culture with tubacin, SAHA, etoposide, and the combinations of tubacin with

Techniques Used: Western Blot

Tubacin enhances cell death induced by SAHA, etoposide, or doxorubicin in transformed LNCaP and MCF-7 cells. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in LNCaP cells cultured for 24
Figure Legend Snippet: Tubacin enhances cell death induced by SAHA, etoposide, or doxorubicin in transformed LNCaP and MCF-7 cells. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in LNCaP cells cultured for 24

Techniques Used: Transformation Assay, Western Blot, Cell Culture

HFS cells are resistant to cell death induced by tubacin cultured in combination with SAHA, etoposide, or doxorubicin. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in HFS cells cultured
Figure Legend Snippet: HFS cells are resistant to cell death induced by tubacin cultured in combination with SAHA, etoposide, or doxorubicin. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in HFS cells cultured

Techniques Used: Cell Culture, Western Blot

Activation of the intrinsic apoptotic pathway is enhanced in transformed cells cultured with tubacin in combination with SAHA or etoposide. ( A ) Western blot analysis showing PARP degradation in LNCaP cells cultured with tubacin, SAHA, or simultaneous
Figure Legend Snippet: Activation of the intrinsic apoptotic pathway is enhanced in transformed cells cultured with tubacin in combination with SAHA or etoposide. ( A ) Western blot analysis showing PARP degradation in LNCaP cells cultured with tubacin, SAHA, or simultaneous

Techniques Used: Activation Assay, Transformation Assay, Cell Culture, Western Blot

Down-regulation of HDAC6 expression in LNCaP cells results in increased sensitivity to cell death induced by SAHA, doxorubicin, or etoposide. ( A ) Western blot analysis in LNCaP cells expressing shRNAs targeting two different sequences within HDAC6 (HDAC6
Figure Legend Snippet: Down-regulation of HDAC6 expression in LNCaP cells results in increased sensitivity to cell death induced by SAHA, doxorubicin, or etoposide. ( A ) Western blot analysis in LNCaP cells expressing shRNAs targeting two different sequences within HDAC6 (HDAC6

Techniques Used: Expressing, Western Blot

51) Product Images from "DNA Damage-Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pHManipulating Cellular pH Suggests Novel Anticancer Therapy"

Article Title: DNA Damage-Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pHManipulating Cellular pH Suggests Novel Anticancer Therapy

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0050001

DNA Damage–Induced Bcl-x L Deamidation Is Mitochondrial Apoptosis–Independent (A) Wild-type thymocytes were pre-incubated with or without Z-VAD-fmk (200 μM), and were then cultured with or without etoposide for 24 h, harvested, and apoptosis was measured by measuring the sub-G1 peak by flow cytometry. The histograms (right panel) represent mean values ± SD ( n = 3). (B) Aliquots of the cells from (A) incubated in the presence or absence of Z-VAD-fmk (200 μM) were analysed for the expression of Bcl-x L and tubulin (as loading control) by immunoblotting. The upper and lower bands of Bcl-x L were quantified and expressed as a percentage of total Bcl-x L . The percentages shown below each lane are means ± SD ( n = 3). (C) Plasmids of shRNA Bax (GFP) and shRNA Bak (DsRed) were cotransfected into purified DN thymocytes using an Amaxa nucleofactor kit. 48 h later, GFP + DsRed + cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture. DN thymocytes transfected with negative control plasmids were treated in parallel. Cells were then processed for immunoblotting with Bcl-x L antibody. The immunoblot was reprobed for Bax and Bak to check the efficiency of gene knockdown. Tubulin was also reprobed as a loading control.
Figure Legend Snippet: DNA Damage–Induced Bcl-x L Deamidation Is Mitochondrial Apoptosis–Independent (A) Wild-type thymocytes were pre-incubated with or without Z-VAD-fmk (200 μM), and were then cultured with or without etoposide for 24 h, harvested, and apoptosis was measured by measuring the sub-G1 peak by flow cytometry. The histograms (right panel) represent mean values ± SD ( n = 3). (B) Aliquots of the cells from (A) incubated in the presence or absence of Z-VAD-fmk (200 μM) were analysed for the expression of Bcl-x L and tubulin (as loading control) by immunoblotting. The upper and lower bands of Bcl-x L were quantified and expressed as a percentage of total Bcl-x L . The percentages shown below each lane are means ± SD ( n = 3). (C) Plasmids of shRNA Bax (GFP) and shRNA Bak (DsRed) were cotransfected into purified DN thymocytes using an Amaxa nucleofactor kit. 48 h later, GFP + DsRed + cells were purified by flow cytometry and treated with etoposide (Etop, 25 μM) for 30 h or exposed to irradiation (IR, 5 Gy) followed by 30 h in culture. DN thymocytes transfected with negative control plasmids were treated in parallel. Cells were then processed for immunoblotting with Bcl-x L antibody. The immunoblot was reprobed for Bax and Bak to check the efficiency of gene knockdown. Tubulin was also reprobed as a loading control.

Techniques Used: Incubation, Cell Culture, Flow Cytometry, Cytometry, Expressing, shRNA, Purification, Irradiation, Transfection, Negative Control

DNA Damage Induces NHE-1 Expression, and Enforced Alkalinisation Promotes Apoptosis of Human B-CLL cells (A) Enforced alkalinisation of cancer cells from patients ( n = 10) with B-CLL causes Bcl-x L deamidation and associated cell death. Treatment with etoposide (Etop) in vitro further amplifies cell death. Patients' cells (PBMC, in the range 85%–95% CD19 + B220 + ) were incubated at pH e values of 7.2, 8.0, or 8.5, and the pH i values were monitored by SNARF-1 staining using flow cytometry. Apoptosis was evaluated by measurement of sub-G1 peaks using flow cytometry. The data shows pooled results from ten patients via 30 values per treatment condition: due to identical values, some symbols overlap. The correlation coefficients ( r ) of deamidation or sub-G1 versus pH i are shown for each treatment. The p value (significance) for each correlation is shown in parentheses. The correlation coefficients of sub-G1 versus deamidation are r = 0.92 ( p
Figure Legend Snippet: DNA Damage Induces NHE-1 Expression, and Enforced Alkalinisation Promotes Apoptosis of Human B-CLL cells (A) Enforced alkalinisation of cancer cells from patients ( n = 10) with B-CLL causes Bcl-x L deamidation and associated cell death. Treatment with etoposide (Etop) in vitro further amplifies cell death. Patients' cells (PBMC, in the range 85%–95% CD19 + B220 + ) were incubated at pH e values of 7.2, 8.0, or 8.5, and the pH i values were monitored by SNARF-1 staining using flow cytometry. Apoptosis was evaluated by measurement of sub-G1 peaks using flow cytometry. The data shows pooled results from ten patients via 30 values per treatment condition: due to identical values, some symbols overlap. The correlation coefficients ( r ) of deamidation or sub-G1 versus pH i are shown for each treatment. The p value (significance) for each correlation is shown in parentheses. The correlation coefficients of sub-G1 versus deamidation are r = 0.92 ( p

Techniques Used: Expressing, In Vitro, Incubation, Staining, Flow Cytometry, Cytometry

Deamidation Disrupts the Sequestration of BH3-Only Proteins by Bcl-x L (A) Bim binds to the native (Asn-Asn) but not deamidated forms of Bcl-x L . Wild-type (C57BL/6) thymocytes (1.5 × 10 7 ) were exposed to 5 Gy irradiation (IR) and then maintained in culture for the times shown, after which cells were lysed and either separated as whole cell lysates (WCL) or as Bim immunoprecipitates, followed by immunoblotting for either Bcl-x L or for Bim. Bim migrates as “extra-long” (EL) or “long” (L) forms. (B) Bcl-x L was immunoprecipitated from lysates derived from purified DN thymocytes treated with/without etoposide (ut/E), followed by immunoblotting for Bim or Puma. The asterisk indicates the light chain of the Bcl-x L antibody used for immunoprecipitation. (C) Anion exchange chromatography of purified rBcl-x L . Sample A was untreated; samples B and C were exposed to pH 8.8 at 37 °C for 2 h and 20 h, respectively. The Figure illustrates superimposed elution profiles for each sample. Peaks A, B, and C had molecular masses of 25, 015.6; 25, 016.4, and 25, 017.2, respectively. (D) Bim binds to native but not to deamidated rBcl-x L . The three different forms of Bcl-x L (A, B, and C) purified by anion-exchange column chromatography shown in (C) were incubated in wild-type thymic lysates (1.5 × 10 7 cell equivalents) at 4 °C for 2 h and then precipitated using nickel beads. The precipitated products were immunoblotted for Bim and Bcl-x L . Quantification of the Bim-L/Bcl-x L ratios ± SD from three independent experiments is shown in the histogram, with the lane A ratio normalised to 1 (*). (E) Primary thymocytes were retrovirally transduced with empty vector or Bcl-x L constructs (wild-type, N52A-N66A, or N52D-N66D). Bcl-x L was immunoprecipitated from lysates derived from 1.5 × 10 6 sorted GFP-positive cells per lane, followed by immunoblotting for Bim or Puma. Note that in the vector lane, at this exposure endogenous Bcl-x L is not visible because of the small number of cells used. The asterisk indicates the light chain of the Bcl-x L antibody used for immunoprecipitation. (F) Peptides SDVEENRTEAPEGTESEMETPSAINGNPSW (peptide 1) and HLADSPAVNGATGHSSSL (peptide 2), and the corresponding deamidated forms, containing the putative deamidation sites N52 and N66, respectively, were generated by digestion of rBcl-x L with chymotrypsin. The chromatographic conditions used for the separation of the peptides in the LC-MS analyses were optimised so as to resolve the Asn, Asp, and iso-Asp forms of peptides 1 and 2. The Asp and iso-Asp forms of the two peptides were identified by spiking an aliquot of a digestion mixture with Asp- or iso-Asp–containing synthetic peptides prior to LC-MS ( Figure S3 ). The chromatograms show LC-MS analyses at time point 72 h of the rBcl-xl base treatment. For both peptides, the major deamidation product is the iso-Asp form; the iso-Asp:Asp ratios are approximately 10:1 for N52 and 5:1 for N66. The unknown peak 3 in peptide 2 could be an isomer of peak 2 or peak 4.
Figure Legend Snippet: Deamidation Disrupts the Sequestration of BH3-Only Proteins by Bcl-x L (A) Bim binds to the native (Asn-Asn) but not deamidated forms of Bcl-x L . Wild-type (C57BL/6) thymocytes (1.5 × 10 7 ) were exposed to 5 Gy irradiation (IR) and then maintained in culture for the times shown, after which cells were lysed and either separated as whole cell lysates (WCL) or as Bim immunoprecipitates, followed by immunoblotting for either Bcl-x L or for Bim. Bim migrates as “extra-long” (EL) or “long” (L) forms. (B) Bcl-x L was immunoprecipitated from lysates derived from purified DN thymocytes treated with/without etoposide (ut/E), followed by immunoblotting for Bim or Puma. The asterisk indicates the light chain of the Bcl-x L antibody used for immunoprecipitation. (C) Anion exchange chromatography of purified rBcl-x L . Sample A was untreated; samples B and C were exposed to pH 8.8 at 37 °C for 2 h and 20 h, respectively. The Figure illustrates superimposed elution profiles for each sample. Peaks A, B, and C had molecular masses of 25, 015.6; 25, 016.4, and 25, 017.2, respectively. (D) Bim binds to native but not to deamidated rBcl-x L . The three different forms of Bcl-x L (A, B, and C) purified by anion-exchange column chromatography shown in (C) were incubated in wild-type thymic lysates (1.5 × 10 7 cell equivalents) at 4 °C for 2 h and then precipitated using nickel beads. The precipitated products were immunoblotted for Bim and Bcl-x L . Quantification of the Bim-L/Bcl-x L ratios ± SD from three independent experiments is shown in the histogram, with the lane A ratio normalised to 1 (*). (E) Primary thymocytes were retrovirally transduced with empty vector or Bcl-x L constructs (wild-type, N52A-N66A, or N52D-N66D). Bcl-x L was immunoprecipitated from lysates derived from 1.5 × 10 6 sorted GFP-positive cells per lane, followed by immunoblotting for Bim or Puma. Note that in the vector lane, at this exposure endogenous Bcl-x L is not visible because of the small number of cells used. The asterisk indicates the light chain of the Bcl-x L antibody used for immunoprecipitation. (F) Peptides SDVEENRTEAPEGTESEMETPSAINGNPSW (peptide 1) and HLADSPAVNGATGHSSSL (peptide 2), and the corresponding deamidated forms, containing the putative deamidation sites N52 and N66, respectively, were generated by digestion of rBcl-x L with chymotrypsin. The chromatographic conditions used for the separation of the peptides in the LC-MS analyses were optimised so as to resolve the Asn, Asp, and iso-Asp forms of peptides 1 and 2. The Asp and iso-Asp forms of the two peptides were identified by spiking an aliquot of a digestion mixture with Asp- or iso-Asp–containing synthetic peptides prior to LC-MS ( Figure S3 ). The chromatograms show LC-MS analyses at time point 72 h of the rBcl-xl base treatment. For both peptides, the major deamidation product is the iso-Asp form; the iso-Asp:Asp ratios are approximately 10:1 for N52 and 5:1 for N66. The unknown peak 3 in peptide 2 could be an isomer of peak 2 or peak 4.

Techniques Used: Irradiation, Immunoprecipitation, Derivative Assay, Purification, Chromatography, Column Chromatography, Incubation, Transduction, Plasmid Preparation, Construct, Generated, Liquid Chromatography with Mass Spectroscopy

Bcl-x L Deamidation Induced by DNA Damage Involves Up-Regulation of the NHE-1 Na/H Antiport (A) Bcl-x L deamidation induced by DNA damage requires de novo protein synthesis. Wild-type thymocytes were either treated with etoposide for 24 h (Etop), or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 24 h, with or without 0.5 μM cycloheximide (CHX). Cell lysates were processed by immunoblotting for Bcl-x L or β-actin (loading control). (B) DNA damage causes up-regulation of NHE-1 in wild-type but not in CD45 −/− Lck F505 thymocytes. Wild-type or CD45 −/− Lck F505 thymocytes were either treated with etoposide (Etop) for 5 h, or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 5 h before immunoblotting for NHE-1 or tubulin (loading control). The histogram shows the quantification of relative NHE-1 expression levels SD from five independent experiments. Lane 3 was defined as 1 (*). (C) Migri-NHE-1 or empty Migri vector were transduced into wild-type or pretumourigenic CD45 −/− Lck F505 thymocytes. 72 h after the first round of infection, cells were immunoblotted for NHE-1 and Bcl-x L . NHE-1 expression levels (NHE-1 relative intensity) were normalised for loading using tubulin values. Deamidation was calculated as in Figure 1 B. The lower left FACS histogram shows the infection efficiency for nontransfected (non), empty-vector transfected (vector), or NHE-1 transfected (NHE-1) cells as percentage GFP-positive cells. The lower right histograms show the mean pH i and apoptosis (sub-G1) values ± SD ( n = 5) analysed on GFP-negative and positive cells. (D) The NHE-1 inhibitor DMA blocks DNA damage-induced alkalinisation (top left panel), Bcl-x L deamidation (lower panel) and apoptosis (top right panel) in wild-type thymocytes. Thymocytes were treated with Etoposide for 24 h, or exposed to 5 Gy of irradiation and then maintained in culture for 24 h, with or without 200 μM DMA. pH i was measured by FACS on live CD4 − CD8 − cells, and the sub-G1 peak was analysed by FACS on CD4 − CD8 − cells to assess apoptosis. The histograms represent mean values ± SD ( n = 3).
Figure Legend Snippet: Bcl-x L Deamidation Induced by DNA Damage Involves Up-Regulation of the NHE-1 Na/H Antiport (A) Bcl-x L deamidation induced by DNA damage requires de novo protein synthesis. Wild-type thymocytes were either treated with etoposide for 24 h (Etop), or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 24 h, with or without 0.5 μM cycloheximide (CHX). Cell lysates were processed by immunoblotting for Bcl-x L or β-actin (loading control). (B) DNA damage causes up-regulation of NHE-1 in wild-type but not in CD45 −/− Lck F505 thymocytes. Wild-type or CD45 −/− Lck F505 thymocytes were either treated with etoposide (Etop) for 5 h, or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 5 h before immunoblotting for NHE-1 or tubulin (loading control). The histogram shows the quantification of relative NHE-1 expression levels SD from five independent experiments. Lane 3 was defined as 1 (*). (C) Migri-NHE-1 or empty Migri vector were transduced into wild-type or pretumourigenic CD45 −/− Lck F505 thymocytes. 72 h after the first round of infection, cells were immunoblotted for NHE-1 and Bcl-x L . NHE-1 expression levels (NHE-1 relative intensity) were normalised for loading using tubulin values. Deamidation was calculated as in Figure 1 B. The lower left FACS histogram shows the infection efficiency for nontransfected (non), empty-vector transfected (vector), or NHE-1 transfected (NHE-1) cells as percentage GFP-positive cells. The lower right histograms show the mean pH i and apoptosis (sub-G1) values ± SD ( n = 5) analysed on GFP-negative and positive cells. (D) The NHE-1 inhibitor DMA blocks DNA damage-induced alkalinisation (top left panel), Bcl-x L deamidation (lower panel) and apoptosis (top right panel) in wild-type thymocytes. Thymocytes were treated with Etoposide for 24 h, or exposed to 5 Gy of irradiation and then maintained in culture for 24 h, with or without 200 μM DMA. pH i was measured by FACS on live CD4 − CD8 − cells, and the sub-G1 peak was analysed by FACS on CD4 − CD8 − cells to assess apoptosis. The histograms represent mean values ± SD ( n = 3).

Techniques Used: Irradiation, Expressing, Plasmid Preparation, Infection, FACS, Transfection

NHE-1 Knockdown Blocks DNA Damage–Induced Bcl-x L Deamidation and Apoptosis (A) Empty vector, negative control, or NHE-1shRNA2 were transduced into wild-type thymocytes, then treated with Etoposide (Etop) or irradiation (IR) prior to immunoblotting for NHE-1 and Bcl-x L . (B) Aliquots of the cells from (A) were analysed for pH i . The histogram represents mean values ± SD ( n = 3). (C) Aliquots of the cells from (A) were analysed for apoptosis by Annexin V/PI staining using flow cytometry, as illustrated in a representative experiment (total n = 5). The numbers shown are the percentage of cells in each quandrant. Histograms summarising the percentage of apoptotic cells (Annexin V + PI − ) and dead cells (Annexin V + PI + ) are shown in Figure 6 B.
Figure Legend Snippet: NHE-1 Knockdown Blocks DNA Damage–Induced Bcl-x L Deamidation and Apoptosis (A) Empty vector, negative control, or NHE-1shRNA2 were transduced into wild-type thymocytes, then treated with Etoposide (Etop) or irradiation (IR) prior to immunoblotting for NHE-1 and Bcl-x L . (B) Aliquots of the cells from (A) were analysed for pH i . The histogram represents mean values ± SD ( n = 3). (C) Aliquots of the cells from (A) were analysed for apoptosis by Annexin V/PI staining using flow cytometry, as illustrated in a representative experiment (total n = 5). The numbers shown are the percentage of cells in each quandrant. Histograms summarising the percentage of apoptotic cells (Annexin V + PI − ) and dead cells (Annexin V + PI + ) are shown in Figure 6 B.

Techniques Used: Plasmid Preparation, Negative Control, Irradiation, Staining, Flow Cytometry, Cytometry

DNA Damage Causes Intracellular Alkalinisation and Subsequent Bcl-x L Deamidation (A) Intracellular alkalinisation occurs following DNA damage in wild-type but not in pretumourigenic CD45 −/− Lck F505 thymocytes. Cells were treated with etoposide (Etop) for 20 h or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 20 h. pH i was measured using SNARF by FACS in the gated live CD4 − CD8 − subset. The histograms represent mean values ± SD ( n = 5). (B) Enforced intracellular thymic alkalinisation causes Bcl-x L deamidation. Wild-type thymocytes were maintained in RPMI-1640/10% bovine fetal calf serum buffered at the indicated pH with Tris-HCl for 20 h in the presence of 20 μM monensin prior to lysis and immunoblotting for Bcl-x L . To minimize any deamidation produced during the gel-running process, the resolving gel buffer was adjusted to pH 8.0 in this experiment. The mean ratio of the lower band (native Bcl-x L ) or upper band (deamidated Bcl-x L ) to the total (upper plus lower bands) is shown in the graph (lower panel). The error bars represent SD ( n = 3). Note that deamidation becomes prominent at pH 7.5. (C) Aliquots of the cells from Figure 1 A incubated in the presence or absence of Z-VAD-fmk (200 μM) were analysed for pH i , The histograms represent mean values ±SD ( n = 3). (D) Wild-type or CD45 −/− Lck F505 pretumourigenic thymocytes were cultured for 24 h in media at the pH shown without monensin, with or without etoposide, and then analysed for Bcl-x L deamidation by immunoblotting. The upper and lower bands were quantified and the percentage of upper bands in total Bcl-x L calculated. The percentages shown below each lane are means ± SD ( n = 5). (E) Aliquots of cells used in (D) were assessed for pH i by FACS. The histograms show the pH i of live gated CD4 − CD8 − thymocytes from five independent experiments ±SD. The pH e values refer to the pH values of the extracellular media. (F) Apoptosis of aliquots of the cells from (D) was analysed by FACS. The histogram shows the sub-G1 peak (%) of CD4 − CD8 − thymocytes from five independent experiments ± SD. (G) Wild-type (wt), N52A-N66A (AA), N52D-N66D (DD) Bcl-x L , and empty vector were retrovirally transduced into thymocytes. GFP-positive cells were FACS sorted (left panel) and cultured in media with the pH e shown for 24 h or 48 h, then processed for immunoblotting with Bcl-x L antibody (middle panel). Note that 8 × 10 6 and 1 × 10 6 cell equivalents were loaded per lane for the empty vector (lanes 1–3) and Bcl-x L (lanes 4–12) transfectants, respectively, such that the endogenous Bcl-x L is invisible in lanes 4–12. The histogram (right panel) shows mean apoptosis (sub-G1) values ±SD generated from five independent experiments.
Figure Legend Snippet: DNA Damage Causes Intracellular Alkalinisation and Subsequent Bcl-x L Deamidation (A) Intracellular alkalinisation occurs following DNA damage in wild-type but not in pretumourigenic CD45 −/− Lck F505 thymocytes. Cells were treated with etoposide (Etop) for 20 h or exposed to 5 Gy of irradiation (IR) and then maintained in culture for 20 h. pH i was measured using SNARF by FACS in the gated live CD4 − CD8 − subset. The histograms represent mean values ± SD ( n = 5). (B) Enforced intracellular thymic alkalinisation causes Bcl-x L deamidation. Wild-type thymocytes were maintained in RPMI-1640/10% bovine fetal calf serum buffered at the indicated pH with Tris-HCl for 20 h in the presence of 20 μM monensin prior to lysis and immunoblotting for Bcl-x L . To minimize any deamidation produced during the gel-running process, the resolving gel buffer was adjusted to pH 8.0 in this experiment. The mean ratio of the lower band (native Bcl-x L ) or upper band (deamidated Bcl-x L ) to the total (upper plus lower bands) is shown in the graph (lower panel). The error bars represent SD ( n = 3). Note that deamidation becomes prominent at pH 7.5. (C) Aliquots of the cells from Figure 1 A incubated in the presence or absence of Z-VAD-fmk (200 μM) were analysed for pH i , The histograms represent mean values ±SD ( n = 3). (D) Wild-type or CD45 −/− Lck F505 pretumourigenic thymocytes were cultured for 24 h in media at the pH shown without monensin, with or without etoposide, and then analysed for Bcl-x L deamidation by immunoblotting. The upper and lower bands were quantified and the percentage of upper bands in total Bcl-x L calculated. The percentages shown below each lane are means ± SD ( n = 5). (E) Aliquots of cells used in (D) were assessed for pH i by FACS. The histograms show the pH i of live gated CD4 − CD8 − thymocytes from five independent experiments ±SD. The pH e values refer to the pH values of the extracellular media. (F) Apoptosis of aliquots of the cells from (D) was analysed by FACS. The histogram shows the sub-G1 peak (%) of CD4 − CD8 − thymocytes from five independent experiments ± SD. (G) Wild-type (wt), N52A-N66A (AA), N52D-N66D (DD) Bcl-x L , and empty vector were retrovirally transduced into thymocytes. GFP-positive cells were FACS sorted (left panel) and cultured in media with the pH e shown for 24 h or 48 h, then processed for immunoblotting with Bcl-x L antibody (middle panel). Note that 8 × 10 6 and 1 × 10 6 cell equivalents were loaded per lane for the empty vector (lanes 1–3) and Bcl-x L (lanes 4–12) transfectants, respectively, such that the endogenous Bcl-x L is invisible in lanes 4–12. The histogram (right panel) shows mean apoptosis (sub-G1) values ±SD generated from five independent experiments.

Techniques Used: Irradiation, FACS, Lysis, Produced, Incubation, Cell Culture, Plasmid Preparation, Generated

52) Product Images from "New role for nuclear hormone receptors and coactivators in regulation of BRCA1-mediated DNA repair in breast cancer cell lines"

Article Title: New role for nuclear hormone receptors and coactivators in regulation of BRCA1-mediated DNA repair in breast cancer cell lines

Journal: Breast Cancer Research

doi: 10.1186/bcr1362

BRCA1 inhibition decreases DNA damage repair and cell survival in human breast cancer cell lines. (a) Expression of dominant-negative BRCA1 in human breast cancer cell lines. T47D and MDA-MB-468 cells were stably transfected with the BRCA1 carboxyl-terminal truncation mutant (BRCA1delC) or neomycin expression vector (neo). Protein extracts from these clones were subjected to western blotting with anti-BRCA1 antibody. Anti-β-actin antibody was used to determine relative amounts of protein in each lane. Representative blots are shown. (b) BRCA1 inhibition decreases the expression of double-strand break repair proteins in human breast cancer cell lines. T47D or MDA-MB-468 BRCA1delC or neomycin-resistant control (neo) clones were treated with etoposide (etopo) or vehicle prior to western blotting with antibodies indicated at the left. Blots were stripped and incubated with anti-β-actin antibody to determine relative amounts of protein in each lane. Representative blots are shown. (c) T47D and MDA-MB-468 BRCA1delC clones exhibit increased DNA damage when treated with etoposide. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2RA alone or in combination (E2/RA) before exposure to etoposide. Vehicle-treated cultures were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. Error bars indicate SEM. (d) T47D and MDA-MB-468 BRCA1delC clones exhibit decreased DNA repair activity. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. Error bars indicate SEM. (e) Increased cell survival in etoposide-treated T47D and MDA-MB-468 BRCA1delC clones. Neomycin control clones were used as controls. Cultures were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). Cell death was quantified by TdT-mediated dUTP nick end labelling assay. These experiments were repeated three times with similar results. Error bars indicate SEM. (f) No complex formation of BRCA1delC with CBP and estrogen receptor α (ERα) in T47D and MDA-MB-468 cells. Stable clones expressing BRCA1delC were treated with E2 or RA as described in Materials and Methods. Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. In contrast, both wild-type and mutant BRCA1 immunoprecipitated from both cell lines with anti-BRCA1 antibody (lower panel). These experiments were performed three times with similar results; representative blots are shown.
Figure Legend Snippet: BRCA1 inhibition decreases DNA damage repair and cell survival in human breast cancer cell lines. (a) Expression of dominant-negative BRCA1 in human breast cancer cell lines. T47D and MDA-MB-468 cells were stably transfected with the BRCA1 carboxyl-terminal truncation mutant (BRCA1delC) or neomycin expression vector (neo). Protein extracts from these clones were subjected to western blotting with anti-BRCA1 antibody. Anti-β-actin antibody was used to determine relative amounts of protein in each lane. Representative blots are shown. (b) BRCA1 inhibition decreases the expression of double-strand break repair proteins in human breast cancer cell lines. T47D or MDA-MB-468 BRCA1delC or neomycin-resistant control (neo) clones were treated with etoposide (etopo) or vehicle prior to western blotting with antibodies indicated at the left. Blots were stripped and incubated with anti-β-actin antibody to determine relative amounts of protein in each lane. Representative blots are shown. (c) T47D and MDA-MB-468 BRCA1delC clones exhibit increased DNA damage when treated with etoposide. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2RA alone or in combination (E2/RA) before exposure to etoposide. Vehicle-treated cultures were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. Error bars indicate SEM. (d) T47D and MDA-MB-468 BRCA1delC clones exhibit decreased DNA repair activity. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. Error bars indicate SEM. (e) Increased cell survival in etoposide-treated T47D and MDA-MB-468 BRCA1delC clones. Neomycin control clones were used as controls. Cultures were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). Cell death was quantified by TdT-mediated dUTP nick end labelling assay. These experiments were repeated three times with similar results. Error bars indicate SEM. (f) No complex formation of BRCA1delC with CBP and estrogen receptor α (ERα) in T47D and MDA-MB-468 cells. Stable clones expressing BRCA1delC were treated with E2 or RA as described in Materials and Methods. Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. In contrast, both wild-type and mutant BRCA1 immunoprecipitated from both cell lines with anti-BRCA1 antibody (lower panel). These experiments were performed three times with similar results; representative blots are shown.

Techniques Used: Inhibition, Expressing, Dominant Negative Mutation, Multiple Displacement Amplification, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, Clone Assay, Western Blot, Incubation, Negative Control, Activity Assay, Immunoprecipitation

E2 inhibits and RA enhances DNA damage-mediated apoptosis in human breast cancer cell lines. (a) Estrogen receptor (ER)-positive human breast cancer cell lines (MCF7 and T47D) and ER-negative lines (MDA-MB-231 and MDA-MB-468) were treated with 17β-estradiol (E2) or all- trans retinoic acid (RA) alone or in combination (E2/RA) prior to etoposide induced DNA damage. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TdT-mediated dUTP nick end labeling (TUNEL) assay. Error bars indicate SEM. (b) The human breast cancer cell lines identified above were treated with E2 or RA alone or in combination (E2/RA)before treatment with 3 Gy of ionizing radiation to induce double-strand DNA breaks. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TUNEL assay. Error bars indicate SEM. (c) The pro-survival effects of E2 were not mediated by MAPK, PKC, phosphoinositide 3-kinase, phospholipase Cγ, or Akt/PKB signaling in human breast cancer cell lines. ER-positive breast cancer cell lines MCF7 and T47D were pretreated with PD98059 (PD), SP600125, (SP), SB203580 (SB), SH5, (Akt/PKB inhibitor), Go6976 (Go), LY294002 (LY), or U73122 (U) as indicated in Materials and Methods. Vehicle-treated cells were used as the negative control (con) for the TUNEL assay. (d) E2 inhibits and RA increases the extent of DNA damage in etoposide-treated human breast cancer cell lines. The indicated ER-positive and ER-negative cell lines were treated with E2 or RA alone or in combination (E2/RA), as described above, before exposure to etoposide. Vehicle-treated cells were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. (e) E2 enhances and RA inhibits DNA double-strand break repair in human breast cancer cell lines. The indicated cell lines were treated with E2 or RA alone or in combination (E2/RA) as described above. Vehicle-treated cells were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. (f) E2 or RA treatment does not affect double-strand break repair protein expression in human breast cancer cell lines. The indicated lines were treated with E2 or RA as described above. Vehicle-treated cells were used as the negative control (con). Protein extracts from treated cells were subjected to western blotting with the anti-human antibodies indicated at the left. (g) Complex formation of BRCA1 with CBP and ERα in E2-treated but not RA-treated T47D cells (upper panel). Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. These experiments were performed three times with similar results; representative blots are shown.
Figure Legend Snippet: E2 inhibits and RA enhances DNA damage-mediated apoptosis in human breast cancer cell lines. (a) Estrogen receptor (ER)-positive human breast cancer cell lines (MCF7 and T47D) and ER-negative lines (MDA-MB-231 and MDA-MB-468) were treated with 17β-estradiol (E2) or all- trans retinoic acid (RA) alone or in combination (E2/RA) prior to etoposide induced DNA damage. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TdT-mediated dUTP nick end labeling (TUNEL) assay. Error bars indicate SEM. (b) The human breast cancer cell lines identified above were treated with E2 or RA alone or in combination (E2/RA)before treatment with 3 Gy of ionizing radiation to induce double-strand DNA breaks. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TUNEL assay. Error bars indicate SEM. (c) The pro-survival effects of E2 were not mediated by MAPK, PKC, phosphoinositide 3-kinase, phospholipase Cγ, or Akt/PKB signaling in human breast cancer cell lines. ER-positive breast cancer cell lines MCF7 and T47D were pretreated with PD98059 (PD), SP600125, (SP), SB203580 (SB), SH5, (Akt/PKB inhibitor), Go6976 (Go), LY294002 (LY), or U73122 (U) as indicated in Materials and Methods. Vehicle-treated cells were used as the negative control (con) for the TUNEL assay. (d) E2 inhibits and RA increases the extent of DNA damage in etoposide-treated human breast cancer cell lines. The indicated ER-positive and ER-negative cell lines were treated with E2 or RA alone or in combination (E2/RA), as described above, before exposure to etoposide. Vehicle-treated cells were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. (e) E2 enhances and RA inhibits DNA double-strand break repair in human breast cancer cell lines. The indicated cell lines were treated with E2 or RA alone or in combination (E2/RA) as described above. Vehicle-treated cells were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. (f) E2 or RA treatment does not affect double-strand break repair protein expression in human breast cancer cell lines. The indicated lines were treated with E2 or RA as described above. Vehicle-treated cells were used as the negative control (con). Protein extracts from treated cells were subjected to western blotting with the anti-human antibodies indicated at the left. (g) Complex formation of BRCA1 with CBP and ERα in E2-treated but not RA-treated T47D cells (upper panel). Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. These experiments were performed three times with similar results; representative blots are shown.

Techniques Used: Multiple Displacement Amplification, Negative Control, End Labeling, TUNEL Assay, Plasmid Preparation, Activity Assay, Expressing, Western Blot, Immunoprecipitation, Incubation

BRCA1 depletion decreases DNA damage repair and cell survival in human breast cancer cell lines. (a) Reduced BRCA1 expression in human breast cancer cell lines transfected with siRNA. T47D and MDA-MB-468 cells were transiently transfected with BRCA1 siRNA or an unrelated siRNA (mock). Protein extracts from these clones were subjected to western blotting with anti-BRCA1 antibody. Anti-β-actin antibody was used to determine relative amounts of protein in each lane. Representative blots are shown. (b) Decreased BRCA1 expression results in increased DNA damage when treated with etoposide. T47D and MDA-MB-468 lines transfected with BRCA1 or unrelated siRNA (mock) were pretreated with 17β-estradiol (E2) or all- trans retinoic acid (RA) alone or in combination (E2/RA) before exposure to etoposide. Vehicle-treated cultures were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. Error bars indicate SEM. (c) Decreased BRCA1 expression results in decreased DNA repair activity. T47D or MDA-MB-468 lines transfected with BRCA1 or unrelated siRNA (mock) were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. Error bars indicate SEM. (d) Decreased BRCA1 expression results in decreased cell survival in etoposide-treated T47D and MDA-MB-468 lines transfected with BRCA1 siRNA. Cultures transfected with unrelated siRNA (mock) were used as controls. Cultures were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). Cell death was quantified by TdT-mediated dUTP nick end labelling assay. These experiments were repeated three times with similar results. Error bars indicate SEM.
Figure Legend Snippet: BRCA1 depletion decreases DNA damage repair and cell survival in human breast cancer cell lines. (a) Reduced BRCA1 expression in human breast cancer cell lines transfected with siRNA. T47D and MDA-MB-468 cells were transiently transfected with BRCA1 siRNA or an unrelated siRNA (mock). Protein extracts from these clones were subjected to western blotting with anti-BRCA1 antibody. Anti-β-actin antibody was used to determine relative amounts of protein in each lane. Representative blots are shown. (b) Decreased BRCA1 expression results in increased DNA damage when treated with etoposide. T47D and MDA-MB-468 lines transfected with BRCA1 or unrelated siRNA (mock) were pretreated with 17β-estradiol (E2) or all- trans retinoic acid (RA) alone or in combination (E2/RA) before exposure to etoposide. Vehicle-treated cultures were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. Error bars indicate SEM. (c) Decreased BRCA1 expression results in decreased DNA repair activity. T47D or MDA-MB-468 lines transfected with BRCA1 or unrelated siRNA (mock) were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. Error bars indicate SEM. (d) Decreased BRCA1 expression results in decreased cell survival in etoposide-treated T47D and MDA-MB-468 lines transfected with BRCA1 siRNA. Cultures transfected with unrelated siRNA (mock) were used as controls. Cultures were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). Cell death was quantified by TdT-mediated dUTP nick end labelling assay. These experiments were repeated three times with similar results. Error bars indicate SEM.

Techniques Used: Expressing, Transfection, Multiple Displacement Amplification, Clone Assay, Western Blot, Negative Control, Activity Assay, Plasmid Preparation

BRCA1delC, but not siRNA, inhibits cell cycle progression in human breast cancer cell lines. (a) Decreased bromodeoxyuridine (BrdU) incorporation in T47D and MDA-MB-468 BRCA1delC (del) clones. Neomycin-resistant clones were used as controls. Separate cultures were transiently transfected with BRCA1 siRNA. Clones were treated with etoposide (E) as described in Materials and Methods. These experiments were performed three times with similar results. Error bars indicate SEM. (b) T47D and MDA-MB-468 BRCA1delC and neomycin-resistant control (neo) clones were treated with etoposide (etopo) or vehicle prior to western blotting with the anti-cell-cycle-regulatory antibodies indicated at the left. Blots were stripped and incubated with anti-β-actin antibody to determine the relative amounts of protein in each lane. Representative blots are shown.
Figure Legend Snippet: BRCA1delC, but not siRNA, inhibits cell cycle progression in human breast cancer cell lines. (a) Decreased bromodeoxyuridine (BrdU) incorporation in T47D and MDA-MB-468 BRCA1delC (del) clones. Neomycin-resistant clones were used as controls. Separate cultures were transiently transfected with BRCA1 siRNA. Clones were treated with etoposide (E) as described in Materials and Methods. These experiments were performed three times with similar results. Error bars indicate SEM. (b) T47D and MDA-MB-468 BRCA1delC and neomycin-resistant control (neo) clones were treated with etoposide (etopo) or vehicle prior to western blotting with the anti-cell-cycle-regulatory antibodies indicated at the left. Blots were stripped and incubated with anti-β-actin antibody to determine the relative amounts of protein in each lane. Representative blots are shown.

Techniques Used: BrdU Incorporation Assay, Multiple Displacement Amplification, Clone Assay, Transfection, Western Blot, Incubation

53) Product Images from "Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress"

Article Title: Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq019

Prep1 overexpression induces a strong apoptotic response in murine cells ( A ; left) Total extracts from murine F9 teratocarcinoma cells stably transfected with a Prep1-expressing or control vector ( 16 ) were resolved in an 8% SDS-PAGE and transferred to a PVDF membrane. The endogenous amount of Prep1 was checked by immunoblotting using a specific monoclonal antibody and normalized to β-actin content. (Middle) Cells were treated with etoposide as indicated or (Right) UV irradiated (UVC 254 nm; 60 J/m 2 ). After 24 h of treatment or 24 h post-UV irradiation the number of AnnexinV-positive (apoptotic) cells was determined by FACS and plotted for overexpressing and control cells. Values are expressed as percentages of total events. C, untreated cells. ( B ) After 12 h of etoposide treatment (as indicated) or 12 h post-UV irradiation (UV C 254 nm; 60 J/m 2 ), total cell extracts from Prep1-overexpressing or control F9 cells were resolved on 12% SDS-PAGE, transferred to a PVDF membrane and active caspases 9 levels were determined by immunoblotting and densitometric analysis, using tubulin for normalization. The densitometric analysis of the blot ( Supplementary Figure S1A ; one of three independent immunoblots is shown as representative experiment) is shown in the graph. ( C ) MEF from Prep1 +/+ and Prep1 i / i embryos infected with a retroviral vector (pBABE) either empty or containing the cDNA for human Prep1 were previously described [( 5 ); Figure 6D]. Infected MEF were treated (or not) with etoposide (200 µM) for 24 h or irradiated with UV light (UV C 254 nm; 60 J/m 2 or 1000 J/m 2 ). Crude extracts were prepared after 24 h of drug treatment or 12 (1000 J/m 2 ) or 24 h (60 J/m 2 ) post-UV irradiation, fractionated in 12% SDS-PAGE, transferred to a PVDF membrane and the level of active caspase-3 determined by densitometric analysis (shown in the graph) of immunoblots ( Supplementary Figure S1B ). The experiment was repeated three times and the results from a representative experiment are shown.
Figure Legend Snippet: Prep1 overexpression induces a strong apoptotic response in murine cells ( A ; left) Total extracts from murine F9 teratocarcinoma cells stably transfected with a Prep1-expressing or control vector ( 16 ) were resolved in an 8% SDS-PAGE and transferred to a PVDF membrane. The endogenous amount of Prep1 was checked by immunoblotting using a specific monoclonal antibody and normalized to β-actin content. (Middle) Cells were treated with etoposide as indicated or (Right) UV irradiated (UVC 254 nm; 60 J/m 2 ). After 24 h of treatment or 24 h post-UV irradiation the number of AnnexinV-positive (apoptotic) cells was determined by FACS and plotted for overexpressing and control cells. Values are expressed as percentages of total events. C, untreated cells. ( B ) After 12 h of etoposide treatment (as indicated) or 12 h post-UV irradiation (UV C 254 nm; 60 J/m 2 ), total cell extracts from Prep1-overexpressing or control F9 cells were resolved on 12% SDS-PAGE, transferred to a PVDF membrane and active caspases 9 levels were determined by immunoblotting and densitometric analysis, using tubulin for normalization. The densitometric analysis of the blot ( Supplementary Figure S1A ; one of three independent immunoblots is shown as representative experiment) is shown in the graph. ( C ) MEF from Prep1 +/+ and Prep1 i / i embryos infected with a retroviral vector (pBABE) either empty or containing the cDNA for human Prep1 were previously described [( 5 ); Figure 6D]. Infected MEF were treated (or not) with etoposide (200 µM) for 24 h or irradiated with UV light (UV C 254 nm; 60 J/m 2 or 1000 J/m 2 ). Crude extracts were prepared after 24 h of drug treatment or 12 (1000 J/m 2 ) or 24 h (60 J/m 2 ) post-UV irradiation, fractionated in 12% SDS-PAGE, transferred to a PVDF membrane and the level of active caspase-3 determined by densitometric analysis (shown in the graph) of immunoblots ( Supplementary Figure S1B ). The experiment was repeated three times and the results from a representative experiment are shown.

Techniques Used: Over Expression, Stable Transfection, Transfection, Expressing, Plasmid Preparation, SDS Page, Irradiation, FACS, Western Blot, Infection

Prep1 overexpression increases Bcl-X L expression ( A ; left) Total RNA was purified from untreated Prep1-overexpressing or control F9 cells, retrotranscribed using polyA + primers and semiquantitative PCR analysis performed with specific primers for murine Bcl-X L and β-actin. The results of the densitometric analysis is shown under each lane. (Right) Crude extracts from the above cells were resolved by 12% SDS-PAGE and transferred to PVDF membrane. Endogenous Bcl-X L protein levels were analyzed by immunoblotting with specific monoclonal antibody and β-actin was used for normalization. Results of the densitometric analysis are shown under each lane. ( B ) Total mRNA and crude extracts were prepared and processed as above for qPCR (experiment performed in triplicate; left) or immunoblotting (right) from cell that were treated (or not) with the etoposide for 12 h. A value of 1 and of 100 was arbitrarily given to Bcl-X L mRNA (left) and protein (right) amount, respectively, in untreated cells infected with the control vector.
Figure Legend Snippet: Prep1 overexpression increases Bcl-X L expression ( A ; left) Total RNA was purified from untreated Prep1-overexpressing or control F9 cells, retrotranscribed using polyA + primers and semiquantitative PCR analysis performed with specific primers for murine Bcl-X L and β-actin. The results of the densitometric analysis is shown under each lane. (Right) Crude extracts from the above cells were resolved by 12% SDS-PAGE and transferred to PVDF membrane. Endogenous Bcl-X L protein levels were analyzed by immunoblotting with specific monoclonal antibody and β-actin was used for normalization. Results of the densitometric analysis are shown under each lane. ( B ) Total mRNA and crude extracts were prepared and processed as above for qPCR (experiment performed in triplicate; left) or immunoblotting (right) from cell that were treated (or not) with the etoposide for 12 h. A value of 1 and of 100 was arbitrarily given to Bcl-X L mRNA (left) and protein (right) amount, respectively, in untreated cells infected with the control vector.

Techniques Used: Over Expression, Expressing, Purification, Polymerase Chain Reaction, SDS Page, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation

Endogenous p53 expression levels increase in Prep1 overexpressing cells and its depletion rescues the apoptotic phenotype. ( A ; left) Total RNA was purified from untreated Prep1-overexpressing or control F9 cells, retrotranscribed using polyA + primers and semiquantitative PCR analysis performed using specific primers for murine p53 and β-actin. Densitometric analysis results are shown under each lane. (Right) Crude extracts from the above cells were fractionated by 12% SDS-PAGE and transferred to PVDF membrane. Endogenous p53 levels were analyzed by immunoblotting with a specific monoclonal antibody. Tubulin was used for normalization. Densitometric analysis results are shown under each lane. ( B ) Total mRNA form ND ( n = 3) and DS ( n = 4) fibroblast lines was purified and processed as above. Quantitative real-time PCR was performed using primers specific for human p53 and the data are normalized to β-actin mRNA values. ( C ) Prep1-overexpressing or control F9 cells were treated with etoposide as shown or irradiated with UV C (254 nm; 60 J/m 2 ). After 12 h of treatment or 12 h post-irradiation, crude extracts were prepared, resolved by 12% SDS-PAGE and transferred in PVDF membrane. The amount of p53 protein was determined by immunoblotting using a specific monoclonal antibody and the value normalized to tubulin. The graph reports the representative results of the above immunoblot (from one of three independent experiments), which was exposed for a short time to maintain p53 in the linear range for most of the lanes and in which p53 is not visible either in the C or 1-µM lanes. However, a longer exposure of the same blot shows the presence of p53 in the relevant C and 1-µM lanes ( Supplementary Figure S1C ). ( D ) Crude extracts were prepared from ND and DS fibroblasts treated (or not) with etoposide for 24 h, as indicated, fractionated and transferred as above. The levels of p53 were determined by densitometric analysis of the immunoblot, performed using specific monoclonal antibody and normalized to tubulin. C, untreated cells. The representative results of one of three independent experiments (using different ND and DS fibroblast lines) are shown. ( E ; left) Prep1 overexpressing F9 cells were infected with p-retro-super vector containing either a p53-specific or scrambled sequence shRNA and the level of endogenous p53 protein was detected from an aliquot of cells by immunoblotting using a monoclonal antibody and quantitated by densitometry and normalized to β-actin content. (Right) Another aliquot of knocked down or control Prep1-overexpressing F9 cells was treated with etoposide (1 µM) for 24 h and apoptotic cells were detected by flow cytometry using Annexin-V staining and their number plotted. The values were expressed as percentage of total events.
Figure Legend Snippet: Endogenous p53 expression levels increase in Prep1 overexpressing cells and its depletion rescues the apoptotic phenotype. ( A ; left) Total RNA was purified from untreated Prep1-overexpressing or control F9 cells, retrotranscribed using polyA + primers and semiquantitative PCR analysis performed using specific primers for murine p53 and β-actin. Densitometric analysis results are shown under each lane. (Right) Crude extracts from the above cells were fractionated by 12% SDS-PAGE and transferred to PVDF membrane. Endogenous p53 levels were analyzed by immunoblotting with a specific monoclonal antibody. Tubulin was used for normalization. Densitometric analysis results are shown under each lane. ( B ) Total mRNA form ND ( n = 3) and DS ( n = 4) fibroblast lines was purified and processed as above. Quantitative real-time PCR was performed using primers specific for human p53 and the data are normalized to β-actin mRNA values. ( C ) Prep1-overexpressing or control F9 cells were treated with etoposide as shown or irradiated with UV C (254 nm; 60 J/m 2 ). After 12 h of treatment or 12 h post-irradiation, crude extracts were prepared, resolved by 12% SDS-PAGE and transferred in PVDF membrane. The amount of p53 protein was determined by immunoblotting using a specific monoclonal antibody and the value normalized to tubulin. The graph reports the representative results of the above immunoblot (from one of three independent experiments), which was exposed for a short time to maintain p53 in the linear range for most of the lanes and in which p53 is not visible either in the C or 1-µM lanes. However, a longer exposure of the same blot shows the presence of p53 in the relevant C and 1-µM lanes ( Supplementary Figure S1C ). ( D ) Crude extracts were prepared from ND and DS fibroblasts treated (or not) with etoposide for 24 h, as indicated, fractionated and transferred as above. The levels of p53 were determined by densitometric analysis of the immunoblot, performed using specific monoclonal antibody and normalized to tubulin. C, untreated cells. The representative results of one of three independent experiments (using different ND and DS fibroblast lines) are shown. ( E ; left) Prep1 overexpressing F9 cells were infected with p-retro-super vector containing either a p53-specific or scrambled sequence shRNA and the level of endogenous p53 protein was detected from an aliquot of cells by immunoblotting using a monoclonal antibody and quantitated by densitometry and normalized to β-actin content. (Right) Another aliquot of knocked down or control Prep1-overexpressing F9 cells was treated with etoposide (1 µM) for 24 h and apoptotic cells were detected by flow cytometry using Annexin-V staining and their number plotted. The values were expressed as percentage of total events.

Techniques Used: Expressing, Purification, Polymerase Chain Reaction, SDS Page, Real-time Polymerase Chain Reaction, Irradiation, Infection, Plasmid Preparation, Sequencing, shRNA, Flow Cytometry, Cytometry, Staining

Down syndrome fibroblasts show increased Prep1 levels and are more prone to genotoxic stress induced apoptosis. ( A ) Total mRNA from normal (ND) and Down syndrome (DS) skin fibroblasts was purified, retro-transcribed using a poly-A + primer, quantitative real-time PCR analysis was performed using primers specific for human Prep1 (‘Material and Methods’ section) and the data normalized to β-actin mRNA values. ( B ) The average of Prep1 protein levels from three ND and five DS lines was calculated from the densitometric analysis of several immunoblots (data not shown). ( C ) ND ( n = 4) and DS ( n = 5) fibroblasts were treated (or not) with etoposide for 24 h. The number Annexin V-positive (i.e. apoptotic) cells was measured by FACS and values were expressed as percentage of total events. ( D ) Levels of active caspase 3 were detected by immunoblot with specific polyclonal antibodies using crude extracts from ND and DS fibroblasts treated (or not) with etoposide for 24 h at the concentrations indicated in the figure. The graph shows the results of the densitometric analysis, normalized to tubulin. C, untreated cells. ( E ) Cultures of three ND and three DS fibroblast lines were divided in two aliquots: one was treated with 200 µM etoposide for 24 h and the level of apoptosis measured by FACS using Annexin V staining and normalized to the level of apoptosis in the relative ND line. The other aliquot was used to determine the level of Prep1 in each line by immunoblot and densitometric analysis and normalized to the Prep1 level of the relative ND line. The relative Prep1 levels were plotted against the relative number of Annexin V positive cells for each DS line. A value of 100 was arbitrarily given to the Prep1 level and the number of Annexin V-positive cells in the three ND lines analyzed; therefore, a single ND value is reported in the graph.
Figure Legend Snippet: Down syndrome fibroblasts show increased Prep1 levels and are more prone to genotoxic stress induced apoptosis. ( A ) Total mRNA from normal (ND) and Down syndrome (DS) skin fibroblasts was purified, retro-transcribed using a poly-A + primer, quantitative real-time PCR analysis was performed using primers specific for human Prep1 (‘Material and Methods’ section) and the data normalized to β-actin mRNA values. ( B ) The average of Prep1 protein levels from three ND and five DS lines was calculated from the densitometric analysis of several immunoblots (data not shown). ( C ) ND ( n = 4) and DS ( n = 5) fibroblasts were treated (or not) with etoposide for 24 h. The number Annexin V-positive (i.e. apoptotic) cells was measured by FACS and values were expressed as percentage of total events. ( D ) Levels of active caspase 3 were detected by immunoblot with specific polyclonal antibodies using crude extracts from ND and DS fibroblasts treated (or not) with etoposide for 24 h at the concentrations indicated in the figure. The graph shows the results of the densitometric analysis, normalized to tubulin. C, untreated cells. ( E ) Cultures of three ND and three DS fibroblast lines were divided in two aliquots: one was treated with 200 µM etoposide for 24 h and the level of apoptosis measured by FACS using Annexin V staining and normalized to the level of apoptosis in the relative ND line. The other aliquot was used to determine the level of Prep1 in each line by immunoblot and densitometric analysis and normalized to the Prep1 level of the relative ND line. The relative Prep1 levels were plotted against the relative number of Annexin V positive cells for each DS line. A value of 100 was arbitrarily given to the Prep1 level and the number of Annexin V-positive cells in the three ND lines analyzed; therefore, a single ND value is reported in the graph.

Techniques Used: Purification, Real-time Polymerase Chain Reaction, Western Blot, FACS, Staining

54) Product Images from "Restoration of ASC expression sensitizes colorectal cancer cells to genotoxic stress-induced caspase-independent cell death"

Article Title: Restoration of ASC expression sensitizes colorectal cancer cells to genotoxic stress-induced caspase-independent cell death

Journal: Cancer letters

doi: 10.1016/j.canlet.2012.12.020

Inflammasome signaling is not required for DNA damage-induced cell death of DLD-1 cells (A) ASC-deficient or ASC-expressing DLD-1 cells were treated with etoposide (75 µM), camptothecin (CPT, 2 µM), or doxorubicin (1.5 µM) for 28 h, and the culture supernatants or cellular lysates were immunoblotted with the indicated antibodies. (B) DLD-1 cells or PMA-primed THP-1 cells were treated with LPS (0.5 µg/ml, 4 h) and nigericin (10 µM, 1 h), or transfected with poly (dA:dT) (1 µg, 6 h). Culture supernatants or lysates were immunoblotted with the indicated antibodies, or assayed for LDH release. (C) DLD-1-ASC cells were treated with etoposide (100 µM, 24 h) and stained with an anti-ASC antibody. Immunofluorescence was then detected as described in Materials and methods.
Figure Legend Snippet: Inflammasome signaling is not required for DNA damage-induced cell death of DLD-1 cells (A) ASC-deficient or ASC-expressing DLD-1 cells were treated with etoposide (75 µM), camptothecin (CPT, 2 µM), or doxorubicin (1.5 µM) for 28 h, and the culture supernatants or cellular lysates were immunoblotted with the indicated antibodies. (B) DLD-1 cells or PMA-primed THP-1 cells were treated with LPS (0.5 µg/ml, 4 h) and nigericin (10 µM, 1 h), or transfected with poly (dA:dT) (1 µg, 6 h). Culture supernatants or lysates were immunoblotted with the indicated antibodies, or assayed for LDH release. (C) DLD-1-ASC cells were treated with etoposide (100 µM, 24 h) and stained with an anti-ASC antibody. Immunofluorescence was then detected as described in Materials and methods.

Techniques Used: Expressing, Cycling Probe Technology, Transfection, Staining, Immunofluorescence

Decreased ASC expression attenuates DNA damage-induced HT-29 or macrophage cell death (A) HT-29 cells were transfected with si-control or si-ASC RNA (50 nM), and after 48 h of transfection, cells were incubated with etoposide as indicated for 30 h and assayed for LDH release. Cellular lysates were immunoblotted with the indicated antibodies. Asterisk indicates significant difference compared to si-ASC-transfected group ( n = 3, * p
Figure Legend Snippet: Decreased ASC expression attenuates DNA damage-induced HT-29 or macrophage cell death (A) HT-29 cells were transfected with si-control or si-ASC RNA (50 nM), and after 48 h of transfection, cells were incubated with etoposide as indicated for 30 h and assayed for LDH release. Cellular lysates were immunoblotted with the indicated antibodies. Asterisk indicates significant difference compared to si-ASC-transfected group ( n = 3, * p

Techniques Used: Expressing, Transfection, Incubation

ASC expression sensitizes human colorectal cancer DLD-1 cells to DNA damaging agents (A) Cellular lysates from various cancer cell lines or mouse bone marrow-derived macrophages (BMDMs) were immunoblotted with anti-ASC or anti-β-actin antibodies. (B) ASC expression in DLD-1 cells with or without 5-AD priming (2 µM, 4 days) was determined by RT-PCR or western blot analysis. (C) DLD-1 cells with or without 5-AD-priming (2 µM, 48 h) were treated with etoposide (50 µM) or doxorubicin (1 µM) for additional 38 h. Cell survival rate was determined by MTT assay. Asterisks indicate significant differences between unprimed and 5-AD-primed cells ( n = 3, p
Figure Legend Snippet: ASC expression sensitizes human colorectal cancer DLD-1 cells to DNA damaging agents (A) Cellular lysates from various cancer cell lines or mouse bone marrow-derived macrophages (BMDMs) were immunoblotted with anti-ASC or anti-β-actin antibodies. (B) ASC expression in DLD-1 cells with or without 5-AD priming (2 µM, 4 days) was determined by RT-PCR or western blot analysis. (C) DLD-1 cells with or without 5-AD-priming (2 µM, 48 h) were treated with etoposide (50 µM) or doxorubicin (1 µM) for additional 38 h. Cell survival rate was determined by MTT assay. Asterisks indicate significant differences between unprimed and 5-AD-primed cells ( n = 3, p

Techniques Used: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, MTT Assay

Etoposide-induced cell death of ASC-expressing colorectal cancer cells is caspase-independent (A) DLD-1-puro or DLD-1-ASC cells were treated with etoposide (75 µM, 24 h) and assayed for annexin V/PI staining by flow cytometry. (B) DLD-1 cells with or without 5-AD priming, DLD-1-puro, and DLD-1-ASC cells were treated with zVAD or YVAD (20 µM, 30 min), followed by treatment with etoposide (50 µM) for 38 h, as indicated. Cell survival was measured by MTT assay. (C) To determine cell survival rate, HT-29 cells were treated with etoposide (100 µM, 36 h) in the presence of zVAD (20 µM, 30 min pretreat), or treated with TNF-α (30 ng/ml, 16 h) after pretreatment with CHX (10 µg/mL) or zVAD (20 µM) for 30 min, as indicated. TC indicates TNF-α plus CHX. For immunoblotting, HT-29 cells were treated with etoposide (100 µM, 24 h) or TNF-α (30 ng/ml, 16 h) after pretreatment with zVAD (20 µM) or cycloheximide (10 µg/ml) for 30 min, as indicated. (D) DLD-1-ASC cells were pretreated with Nec-1 (40 µM) or zVAD (20 µM) for 30 min, and treated with etoposide (100 µM) for 31 h. LDH release was then assayed for determining cell death. Asterisk indicates the significant difference compared to only etoposide-treated cells ( n = 4, * p
Figure Legend Snippet: Etoposide-induced cell death of ASC-expressing colorectal cancer cells is caspase-independent (A) DLD-1-puro or DLD-1-ASC cells were treated with etoposide (75 µM, 24 h) and assayed for annexin V/PI staining by flow cytometry. (B) DLD-1 cells with or without 5-AD priming, DLD-1-puro, and DLD-1-ASC cells were treated with zVAD or YVAD (20 µM, 30 min), followed by treatment with etoposide (50 µM) for 38 h, as indicated. Cell survival was measured by MTT assay. (C) To determine cell survival rate, HT-29 cells were treated with etoposide (100 µM, 36 h) in the presence of zVAD (20 µM, 30 min pretreat), or treated with TNF-α (30 ng/ml, 16 h) after pretreatment with CHX (10 µg/mL) or zVAD (20 µM) for 30 min, as indicated. TC indicates TNF-α plus CHX. For immunoblotting, HT-29 cells were treated with etoposide (100 µM, 24 h) or TNF-α (30 ng/ml, 16 h) after pretreatment with zVAD (20 µM) or cycloheximide (10 µg/ml) for 30 min, as indicated. (D) DLD-1-ASC cells were pretreated with Nec-1 (40 µM) or zVAD (20 µM) for 30 min, and treated with etoposide (100 µM) for 31 h. LDH release was then assayed for determining cell death. Asterisk indicates the significant difference compared to only etoposide-treated cells ( n = 4, * p

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, MTT Assay

Mitochondrial ROS and JNK activation is required for etoposide-induced cell death of ASC-expressing colorectal cancer cells (A) DLD-1-puro or DLD-1-ASC cells were treated with etoposide (100 µM) for the indicated times. Cells were stained with MitoSox and analyzed for mitochondrial ROS production by flow cytometry. (B) DLD-1-ASC cells were treated with etoposide (100 µM, 30 h) in the presence of NAC (2 mM, 30 min pretreat), and assayed for LDH release. Asterisk indicates the significant difference from etoposide-treated cells ( n = 4, * p
Figure Legend Snippet: Mitochondrial ROS and JNK activation is required for etoposide-induced cell death of ASC-expressing colorectal cancer cells (A) DLD-1-puro or DLD-1-ASC cells were treated with etoposide (100 µM) for the indicated times. Cells were stained with MitoSox and analyzed for mitochondrial ROS production by flow cytometry. (B) DLD-1-ASC cells were treated with etoposide (100 µM, 30 h) in the presence of NAC (2 mM, 30 min pretreat), and assayed for LDH release. Asterisk indicates the significant difference from etoposide-treated cells ( n = 4, * p

Techniques Used: Activation Assay, Expressing, Staining, Flow Cytometry, Cytometry

55) Product Images from "d-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species"

Article Title: d-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species

Journal: Life Science Alliance

doi: 10.26508/lsa.201800045

Pharmacological inhibition of DAO impairs DNA damage– and oncogene-induced senescence. (A–D) U2OS (A, C) and HepG2 (B, D) cells treated with etoposide in the presence of 50 or 100 μM CBIO were subjected to SA-β-gal staining (A, B) and colony-formation assay (C, D). (E , F) U2OS (E) and HepG2 (F) cells were treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO, and the expression levels of the indicated proteins were determined by immunoblot analysis. The protein levels relative to the γ-tubulin levels, except for the phosphorylated p53 (p53 pS15), which was normalized to the total p53 levels, were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (G, H) Hs68 cells treated with 0.5 μM etoposide for 7 d in the presence of 50 μM CBIO were subjected to SA-β-gal staining (G) and EdU incorporation assay (H). The percentage of EdU-positive cells (H, left panel) and representative microscopic images (H right panel) are shown. Bars, 50 μm. (I) Hs68 cells treated with 0.5 μM etoposide for 2 d in the presence of 50 μM CBIO were subjected to immunoblot analysis. The protein levels were quantified as in (E, F). (J) WI-38 cells were transfected with pcDNA3-HA containing oncogenic Ras G12V, and the protein expression of RasG12V was confirmed by immunoblot analysis. (K, L) WI-38 cells transfected as in (J) were selected with 300 μg/ml G418 and treated with 50 μM CBIO. After incubation for 8 d, the cells were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; ** P
Figure Legend Snippet: Pharmacological inhibition of DAO impairs DNA damage– and oncogene-induced senescence. (A–D) U2OS (A, C) and HepG2 (B, D) cells treated with etoposide in the presence of 50 or 100 μM CBIO were subjected to SA-β-gal staining (A, B) and colony-formation assay (C, D). (E , F) U2OS (E) and HepG2 (F) cells were treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO, and the expression levels of the indicated proteins were determined by immunoblot analysis. The protein levels relative to the γ-tubulin levels, except for the phosphorylated p53 (p53 pS15), which was normalized to the total p53 levels, were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (G, H) Hs68 cells treated with 0.5 μM etoposide for 7 d in the presence of 50 μM CBIO were subjected to SA-β-gal staining (G) and EdU incorporation assay (H). The percentage of EdU-positive cells (H, left panel) and representative microscopic images (H right panel) are shown. Bars, 50 μm. (I) Hs68 cells treated with 0.5 μM etoposide for 2 d in the presence of 50 μM CBIO were subjected to immunoblot analysis. The protein levels were quantified as in (E, F). (J) WI-38 cells were transfected with pcDNA3-HA containing oncogenic Ras G12V, and the protein expression of RasG12V was confirmed by immunoblot analysis. (K, L) WI-38 cells transfected as in (J) were selected with 300 μg/ml G418 and treated with 50 μM CBIO. After incubation for 8 d, the cells were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; ** P

Techniques Used: Inhibition, Staining, Colony Assay, Expressing, Software, Transfection, Incubation

Riboflavin treatment activates the DAO activity in the absence of DNA damage. (A) U2OS cells transfected with siRNA for RFVT1 were treated with 2 μM etoposide for 7 d, and the expression levels of RFVT1 were determined by qPCR. (B) U2OS cells depleted of RFVT1 were treated with 2 μM etoposide for 7 d and subjected to FAD quantification. The concentrations of FAD per mg protein of the cells are shown. (C, D) U2OS cells were transfected with pcDNA3-HA containing wt- DAO , selected with 800 μg/ml G418, and treated with 50 μM riboflavin and 5 mM d- serine in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (C) and EdU incorporation assay (D). (E) U2OS cells were treated as in (C, D), and the expression levels of IL-6 were determined by qPCR. (F) U2OS cells overexpressing DAO were treated with 50 μM riboflavin and 5 mM d- serine for 7 d and subjected to immunostaining for 53BP1, HA, and Hoechst staining. Representative microscopic images (left) and box plots of the number of 53BP1 foci in HA-DAO–expressing cells (right) are shown. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th and 25th percentiles and the median, respectively. Error bars (whiskers) indicate the 90th and 10th percentiles, respectively. Statistical significance ( P -value) is shown using the t test analysis ( n = 50 cells). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P
Figure Legend Snippet: Riboflavin treatment activates the DAO activity in the absence of DNA damage. (A) U2OS cells transfected with siRNA for RFVT1 were treated with 2 μM etoposide for 7 d, and the expression levels of RFVT1 were determined by qPCR. (B) U2OS cells depleted of RFVT1 were treated with 2 μM etoposide for 7 d and subjected to FAD quantification. The concentrations of FAD per mg protein of the cells are shown. (C, D) U2OS cells were transfected with pcDNA3-HA containing wt- DAO , selected with 800 μg/ml G418, and treated with 50 μM riboflavin and 5 mM d- serine in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (C) and EdU incorporation assay (D). (E) U2OS cells were treated as in (C, D), and the expression levels of IL-6 were determined by qPCR. (F) U2OS cells overexpressing DAO were treated with 50 μM riboflavin and 5 mM d- serine for 7 d and subjected to immunostaining for 53BP1, HA, and Hoechst staining. Representative microscopic images (left) and box plots of the number of 53BP1 foci in HA-DAO–expressing cells (right) are shown. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th and 25th percentiles and the median, respectively. Error bars (whiskers) indicate the 90th and 10th percentiles, respectively. Statistical significance ( P -value) is shown using the t test analysis ( n = 50 cells). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P

Techniques Used: Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Incubation, Staining, Immunostaining

Ectopic expression of wt-DAO, but not the inactive mutant, promotes senescence. (A) U2OS cells were transfected with pcDNA3-HA containing wt- and R199W- DAO , and the protein expression of DAO was confirmed by immunoblot analysis. (B, C) U2OS cells transfected as in (A) were selected with 800 μg/ml G418 and treated with 2 μM etoposide. After incubation for 7 d, the cells were subjected to SA-β-gal staining (B) and EdU incorporation assay (C). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P
Figure Legend Snippet: Ectopic expression of wt-DAO, but not the inactive mutant, promotes senescence. (A) U2OS cells were transfected with pcDNA3-HA containing wt- and R199W- DAO , and the protein expression of DAO was confirmed by immunoblot analysis. (B, C) U2OS cells transfected as in (A) were selected with 800 μg/ml G418 and treated with 2 μM etoposide. After incubation for 7 d, the cells were subjected to SA-β-gal staining (B) and EdU incorporation assay (C). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P

Techniques Used: Expressing, Mutagenesis, Transfection, Incubation, Staining

d- arginine and d- serine enhances the senescence-promoting effect of DAO. (A, B) U2OS cells were transfected with pcDNA3-HA containing wt- DAO , selected with 800 μg/ml G418, and treated with each of seven d- amino acids at 5 mM (except for d- tyrosine which was used at 2.5 mM) in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (A) and EdU incorporation assay (B). (C, D) U2OS cells were transfected as in (A, B) in the presence of d- arginine, d- serine, l -arginine, or l -serine at 5 mM for 7 d and subjected to SA-β-gal staining (C) and EdU incorporation assay (D). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P
Figure Legend Snippet: d- arginine and d- serine enhances the senescence-promoting effect of DAO. (A, B) U2OS cells were transfected with pcDNA3-HA containing wt- DAO , selected with 800 μg/ml G418, and treated with each of seven d- amino acids at 5 mM (except for d- tyrosine which was used at 2.5 mM) in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (A) and EdU incorporation assay (B). (C, D) U2OS cells were transfected as in (A, B) in the presence of d- arginine, d- serine, l -arginine, or l -serine at 5 mM for 7 d and subjected to SA-β-gal staining (C) and EdU incorporation assay (D). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P

Techniques Used: Transfection, Incubation, Staining

Knockdown of DAO inhibits DNA damage–induced senescence. (A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 7 d, and the expression levels of DAO were determined by qPCR. (B) HepG2 cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 10 μM etoposide for 48 h, and the expression levels of DAO were determined by immunoblot analysis. (C, D) U2OS (C) and HepG2 (D) cells depleted of DAO were treated with 2 and 10 μM etoposide for 7 d and 48 h, respectively, and subjected to SA-β-gal staining. The percentage of SA-β-gal–positive cells (C left panel, D) and representative microscopic images (C right panel) are shown. Bars, 50 μm. (E) U2OS cells depleted of DAO were treated with 2 μM etoposide for 7 d and subjected to colony-formation assay. Relative proliferation rate (upper panel) and representative images (lower panel) are shown. (F) U2OS cells treated as in (E) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. Data are mean ± SD ( n = 3 except in (A) where n = 2 independent experiments). Statistical significance is shown using the t test analysis; * P
Figure Legend Snippet: Knockdown of DAO inhibits DNA damage–induced senescence. (A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 7 d, and the expression levels of DAO were determined by qPCR. (B) HepG2 cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 10 μM etoposide for 48 h, and the expression levels of DAO were determined by immunoblot analysis. (C, D) U2OS (C) and HepG2 (D) cells depleted of DAO were treated with 2 and 10 μM etoposide for 7 d and 48 h, respectively, and subjected to SA-β-gal staining. The percentage of SA-β-gal–positive cells (C left panel, D) and representative microscopic images (C right panel) are shown. Bars, 50 μm. (E) U2OS cells depleted of DAO were treated with 2 μM etoposide for 7 d and subjected to colony-formation assay. Relative proliferation rate (upper panel) and representative images (lower panel) are shown. (F) U2OS cells treated as in (E) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. Data are mean ± SD ( n = 3 except in (A) where n = 2 independent experiments). Statistical significance is shown using the t test analysis; * P

Techniques Used: Transfection, Expressing, Real-time Polymerase Chain Reaction, Staining, Colony Assay, Software

DAO enhances DNA damage–induced ROS accumulation, cooperating with PRODH. (A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 2 d, and the ROS level was measured. (B, C) U2OS (B) and HepG2 (C) cells treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO were subjected to ROS assay. (D) U2OS cells transfected with pcDNA3-HA-DAO and treated with 2 μM etoposide were subjected to ROS assay. (E, F) U2OS cells overexpressing DAO were treated with etoposide in the presence of 1 mM NAC for 7 d and subjected to SA-β-gal staining (E) and EdU incorporation assay (F). (G) U2OS cells treated as in (E, F) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (H, I) U2OS cells treated with etoposide in combination with 50 μM CBIO and 1 mM NAC as indicated for 7 d were subjected to SA-β-gal staining (H) and EdU incorporation assay (I). (J) U2OS cells treated as in (H, I) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (K, L) U2OS cells treated as in (H, I) but 5 mM THFA instead of NAC were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). (M) U2OS cells treated as in (K, L) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (N, O) Hs68 cells treated as in (K, L) were subjected to SA-β-gal staining (N) and EdU incorporation assay (O). (P) Hs68 cells treated as in (N, O) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P
Figure Legend Snippet: DAO enhances DNA damage–induced ROS accumulation, cooperating with PRODH. (A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 2 d, and the ROS level was measured. (B, C) U2OS (B) and HepG2 (C) cells treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO were subjected to ROS assay. (D) U2OS cells transfected with pcDNA3-HA-DAO and treated with 2 μM etoposide were subjected to ROS assay. (E, F) U2OS cells overexpressing DAO were treated with etoposide in the presence of 1 mM NAC for 7 d and subjected to SA-β-gal staining (E) and EdU incorporation assay (F). (G) U2OS cells treated as in (E, F) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (H, I) U2OS cells treated with etoposide in combination with 50 μM CBIO and 1 mM NAC as indicated for 7 d were subjected to SA-β-gal staining (H) and EdU incorporation assay (I). (J) U2OS cells treated as in (H, I) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (K, L) U2OS cells treated as in (H, I) but 5 mM THFA instead of NAC were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). (M) U2OS cells treated as in (K, L) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (N, O) Hs68 cells treated as in (K, L) were subjected to SA-β-gal staining (N) and EdU incorporation assay (O). (P) Hs68 cells treated as in (N, O) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). Data are mean ± SD ( n = 3 independent experiments). Statistical significance is shown using the t test analysis; * P

Techniques Used: Transfection, ROS Assay, Staining, Software

56) Product Images from "Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression"

Article Title: Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression

Journal: Scientific Reports

doi: 10.1038/s41598-017-12111-1

Oligo-Fucoidan reduces CCL2 and IL-6 expression. CCL2 and IL-6 mRNA expression levels were examined in the p53 +/+ ( a , c , e ) and p53 −/− ( b , d , f ) cells. Cells treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h were studied ( a , b ). The cells were pre-incubated with or without Oligo-Fucoidan for 6 h before being treated with etoposide for another 18 h ( c , d ). The cells were analyzed after being exposed to etoposide for only 6 h before being treated with Oligo-Fucoidan for another 18 h ( e , f ). The data represent the mean ± SD of three independent experiments. *p
Figure Legend Snippet: Oligo-Fucoidan reduces CCL2 and IL-6 expression. CCL2 and IL-6 mRNA expression levels were examined in the p53 +/+ ( a , c , e ) and p53 −/− ( b , d , f ) cells. Cells treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h were studied ( a , b ). The cells were pre-incubated with or without Oligo-Fucoidan for 6 h before being treated with etoposide for another 18 h ( c , d ). The cells were analyzed after being exposed to etoposide for only 6 h before being treated with Oligo-Fucoidan for another 18 h ( e , f ). The data represent the mean ± SD of three independent experiments. *p

Techniques Used: Expressing, Incubation

Oligo-Fucoidan and p53 cooperate to protect HCT116 cells against genotoxicity. p53, p21 and γ-H2AX expression levels were analyzed after p53 +/+ ( a ) and p53 −/− ( b ) HCT116 cells were exposed to etoposide (40 μM) for 6 h before being treated with Oligo-Fucoidan (200 μg/ml) for different intervals (0–3 days). HCT116 cells were exposed to etoposide (40 μM) for 6 h before being treated with a higher dose of Oligo-Fucoidan (400 μg/ml) for different periods (0–3 days) (c-d). γ-H2AX, p53, p21 and PTEN expression levels were subsequently analyzed ( c ). ATM, Chk1 and Chk2 signaling activation was assessed ( d ). The protein levels were normalized to those β-actin, and the levels of their corresponding controls were set as 1.
Figure Legend Snippet: Oligo-Fucoidan and p53 cooperate to protect HCT116 cells against genotoxicity. p53, p21 and γ-H2AX expression levels were analyzed after p53 +/+ ( a ) and p53 −/− ( b ) HCT116 cells were exposed to etoposide (40 μM) for 6 h before being treated with Oligo-Fucoidan (200 μg/ml) for different intervals (0–3 days). HCT116 cells were exposed to etoposide (40 μM) for 6 h before being treated with a higher dose of Oligo-Fucoidan (400 μg/ml) for different periods (0–3 days) (c-d). γ-H2AX, p53, p21 and PTEN expression levels were subsequently analyzed ( c ). ATM, Chk1 and Chk2 signaling activation was assessed ( d ). The protein levels were normalized to those β-actin, and the levels of their corresponding controls were set as 1.

Techniques Used: Expressing, Activation Assay

Oligo-Fucoidan and p53 cooperate to regulate DNA damage checkpoints. HCT116 cells were treated with PBS, Oligo-Fucoidan (400 μg/ml) and/or etoposide (40 μM) for 48 h. ( a ) The p53 −/− cell cycle profile was characterized. ( b ) Histograms reveal comparisons of the p53 −/− cell cycle profiles under different experimental settings. ( c ) The p53 +/+ cell cycle profile was assessed. ( d ) Histograms display comparisons of the p53 +/+ cell cycle profiles under different treatment conditions. ( e ) The levels of the indicated apoptotic markers (cleaved PARP and active caspase 3) and their intact molecules were examined. ( f ) The molecules responsible for regulating the G1 and G2/M checkpoints were characterized. The data represent the mean ± SD of three independent experiments. *p
Figure Legend Snippet: Oligo-Fucoidan and p53 cooperate to regulate DNA damage checkpoints. HCT116 cells were treated with PBS, Oligo-Fucoidan (400 μg/ml) and/or etoposide (40 μM) for 48 h. ( a ) The p53 −/− cell cycle profile was characterized. ( b ) Histograms reveal comparisons of the p53 −/− cell cycle profiles under different experimental settings. ( c ) The p53 +/+ cell cycle profile was assessed. ( d ) Histograms display comparisons of the p53 +/+ cell cycle profiles under different treatment conditions. ( e ) The levels of the indicated apoptotic markers (cleaved PARP and active caspase 3) and their intact molecules were examined. ( f ) The molecules responsible for regulating the G1 and G2/M checkpoints were characterized. The data represent the mean ± SD of three independent experiments. *p

Techniques Used:

Oligo-Fucoidan suppresses IL-6 and CCL2 production as well as STAT3 and STAT5 activation. ( a ) HCT116 cells were treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h. IL-6 and CCL2 secretion was measured after the cells were incubated in treatment-free and serum-free medium for 48 h. The results represent the mean ± SD of three independent experiments. *p
Figure Legend Snippet: Oligo-Fucoidan suppresses IL-6 and CCL2 production as well as STAT3 and STAT5 activation. ( a ) HCT116 cells were treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h. IL-6 and CCL2 secretion was measured after the cells were incubated in treatment-free and serum-free medium for 48 h. The results represent the mean ± SD of three independent experiments. *p

Techniques Used: Activation Assay, Incubation

Oligo-Fucoidan prevents intrinsic DNA lesions and mitochondrial ROS generation. ( a ) HCT116 cell lines (p53 +/+ and p53 −/− ) were treated with different doses of Oligo-Fucoidan for 48 h. p53, p21 and γ-HAX expression levels were analyzed in the indicated cells. γ-HAX levels were compared between p53 +/+ and p53 −/− cells after Oligo-Fucoidan treatment. ( b ) Mitochondrial superoxide levels were detected by MitoSOX Red, followed by flow cytometry analysis, in cells treated with PBS (MOCK) or Oligo-Fucoidan (400 μg/ml) for 48 h. ( c ) p53, p21 and γ-HAX expression levels were studied in cells exposed to etoposide (40 μM) for different intervals. The protein levels were normalized to those β-actin, and the levels of their corresponding controls were set as 1. ( d ) Mitochondrial superoxide levels were measured after etoposide (40 μM) and Oligo-Fucoidan (400 μg/ml) administration or etoposide treatment alone for 48 h. ( e ) Cell viability was analyzed after the indicated cells being treated with etoposide alone or co-treated with different concentrations of Oligo-Fucoidan for 48 h. The data represent the mean ± SD of three independent experiments. *p
Figure Legend Snippet: Oligo-Fucoidan prevents intrinsic DNA lesions and mitochondrial ROS generation. ( a ) HCT116 cell lines (p53 +/+ and p53 −/− ) were treated with different doses of Oligo-Fucoidan for 48 h. p53, p21 and γ-HAX expression levels were analyzed in the indicated cells. γ-HAX levels were compared between p53 +/+ and p53 −/− cells after Oligo-Fucoidan treatment. ( b ) Mitochondrial superoxide levels were detected by MitoSOX Red, followed by flow cytometry analysis, in cells treated with PBS (MOCK) or Oligo-Fucoidan (400 μg/ml) for 48 h. ( c ) p53, p21 and γ-HAX expression levels were studied in cells exposed to etoposide (40 μM) for d