etoposide  (Selleck Chemicals)

 
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  • 99
    Name:
    Etoposide
    Description:
    Etoposide is a semisynthetic derivative of podophyllotoxin which inhibits DNA synthesis via topoisomerase II inhibition activity
    Catalog Number:
    s1225
    Product Aliases:
    VP-16, VP-16213
    Molecular Weight:
    588.56
    Price:
    None
    Category:
    Topoisomerase inhibitor DNA Damage
    Formula:
    C29H32O13
    Buy from Supplier


    Structured Review

    Selleck Chemicals etoposide
    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, <t>etoposide.</t> Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.
    Etoposide is a semisynthetic derivative of podophyllotoxin which inhibits DNA synthesis via topoisomerase II inhibition activity
    https://www.bioz.com/result/etoposide/product/Selleck Chemicals
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor"

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    Journal: Scientific Reports

    doi: 10.1038/srep14391

    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.
    Figure Legend Snippet: HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.

    Techniques Used:

    Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.
    Figure Legend Snippet: Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.

    Techniques Used: Single Cell Tracking

    2) Product Images from "Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1"

    Article Title: Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1

    Journal: Leukemia

    doi: 10.1038/leu.2015.99

    ABT-199 synergizes strongly with lymphoma chemotherapy agents that affect MCL1 levels ( a ) Doxorubicin vs. dinaciclib activity comparison as in Figure 1a . ( b ) The indicated lines were treated with doxorubicin at the indicated times and concentrations and subjected to western blotting. ( c-e ) Viability and CI vs. Fa after 24 hours’ exposure to doxorubicin ( c ), etoposide ( d ), or cytarabine ( e ) alone or in combination with ABT-199 in Riva, U2932, and VavP- Bcl2/c-MYC murine tumor cells. Viability shown at 500 nM (500 ng/mL for doxorubicin; quadruplicates ± SEM.)
    Figure Legend Snippet: ABT-199 synergizes strongly with lymphoma chemotherapy agents that affect MCL1 levels ( a ) Doxorubicin vs. dinaciclib activity comparison as in Figure 1a . ( b ) The indicated lines were treated with doxorubicin at the indicated times and concentrations and subjected to western blotting. ( c-e ) Viability and CI vs. Fa after 24 hours’ exposure to doxorubicin ( c ), etoposide ( d ), or cytarabine ( e ) alone or in combination with ABT-199 in Riva, U2932, and VavP- Bcl2/c-MYC murine tumor cells. Viability shown at 500 nM (500 ng/mL for doxorubicin; quadruplicates ± SEM.)

    Techniques Used: Activity Assay, Western Blot

    3) Product Images from "Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines"

    Article Title: Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines

    Journal: Targeted Oncology

    doi: 10.1007/s11523-016-0444-7

    Investigation of apoptotic and necrotic cell death mechanisms in UCCs induced by class I HDAC inhibitor 4SC-202. Relative cell viability measured by MTT-assay in VM-CUB1 and UM-UC-3 cells following 4SC-202 treatment (0.5/2.5 μM, 48 h) in combination with Q-VD-OPh (pan-caspase inhibitor) or Necrox-2 (necrosis inhibitor). Etoposide or H 2 O 2 treatments were used as positive controls. The determined significances of the treated cells relate to the DMSO solvent control. Additional significances between the different treatments are shown by brackets
    Figure Legend Snippet: Investigation of apoptotic and necrotic cell death mechanisms in UCCs induced by class I HDAC inhibitor 4SC-202. Relative cell viability measured by MTT-assay in VM-CUB1 and UM-UC-3 cells following 4SC-202 treatment (0.5/2.5 μM, 48 h) in combination with Q-VD-OPh (pan-caspase inhibitor) or Necrox-2 (necrosis inhibitor). Etoposide or H 2 O 2 treatments were used as positive controls. The determined significances of the treated cells relate to the DMSO solvent control. Additional significances between the different treatments are shown by brackets

    Techniques Used: MTT Assay

    4) Product Images from "Vorinostat enhances the cisplatin-mediated anticancer effects in small cell lung cancer cells"

    Article Title: Vorinostat enhances the cisplatin-mediated anticancer effects in small cell lung cancer cells

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2888-7

    Effects of triple combination treatments of vorinostat with cisplatin and etoposide on the viability and apoptosis of SCLC cells. H209 and H146 cells were treated with or without vorinostat in combination with cisplatin ( a vorinostat at 0.8 μM, and cisplatin and etoposide both at 1 μM; b vorinostat at 0.4 μM, cisplatin at 0.2 μM, and etoposide at 0.6 μM) for 24 h. Cell viability was determined using the MTS assay, and data were represented as mean ± SD in triplicate. A significant reduction in cell viability was documented (*, P
    Figure Legend Snippet: Effects of triple combination treatments of vorinostat with cisplatin and etoposide on the viability and apoptosis of SCLC cells. H209 and H146 cells were treated with or without vorinostat in combination with cisplatin ( a vorinostat at 0.8 μM, and cisplatin and etoposide both at 1 μM; b vorinostat at 0.4 μM, cisplatin at 0.2 μM, and etoposide at 0.6 μM) for 24 h. Cell viability was determined using the MTS assay, and data were represented as mean ± SD in triplicate. A significant reduction in cell viability was documented (*, P

    Techniques Used: MTS Assay

    5) Product Images from "Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer"

    Article Title: Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06162-9

    Loss of APC induces chemotherapy resistance in human SCLC cell lines. a Left: knockdown of APC in H1694 cells with two different shAPC constructs (shAPC#1 and shAPC#2); right: activation of WNT signaling as measured by AXIN2 upregulation and TOPFlash reporter activity (signal fold changes) in these cells. Control cells expressed shRNA with scrambled target sequence (shScr). APC and AXIN2 mRNA levels were measured by quantitative PCR (qPCR). Fold change is reported with respect to control cells, and values were compared using unpaired t tests. Each experiment was performed in biological triplicate (TOPFlash assay results for shAPC#2 are shown from n = 5 experiments). b Percentage of H1694 cells surviving etoposide following 72-h treatment. c Left: fold change in etoposide IC50 following APC knockdown in H1694 cells compared to control cells, and right: fold change in etoposide IC50 in APC knockdown (shAPC#2) cells following overexpression of APC or GFP (control). IC50 values were compared using ratio-paired t tests. d Results from Surveyor assay demonstrating genomic alterations in APC (cleavage products indicated by black bar) following CRISPR–Cas9-guided deletion in H82 sgAPC cells and in-frame deletions in APC that were identified through targeted sequencing of the APC sgRNA site. e WNT activation in H82 sgAPC cells as measured by AXIN2 mRNA levels by qPCR ( p
    Figure Legend Snippet: Loss of APC induces chemotherapy resistance in human SCLC cell lines. a Left: knockdown of APC in H1694 cells with two different shAPC constructs (shAPC#1 and shAPC#2); right: activation of WNT signaling as measured by AXIN2 upregulation and TOPFlash reporter activity (signal fold changes) in these cells. Control cells expressed shRNA with scrambled target sequence (shScr). APC and AXIN2 mRNA levels were measured by quantitative PCR (qPCR). Fold change is reported with respect to control cells, and values were compared using unpaired t tests. Each experiment was performed in biological triplicate (TOPFlash assay results for shAPC#2 are shown from n = 5 experiments). b Percentage of H1694 cells surviving etoposide following 72-h treatment. c Left: fold change in etoposide IC50 following APC knockdown in H1694 cells compared to control cells, and right: fold change in etoposide IC50 in APC knockdown (shAPC#2) cells following overexpression of APC or GFP (control). IC50 values were compared using ratio-paired t tests. d Results from Surveyor assay demonstrating genomic alterations in APC (cleavage products indicated by black bar) following CRISPR–Cas9-guided deletion in H82 sgAPC cells and in-frame deletions in APC that were identified through targeted sequencing of the APC sgRNA site. e WNT activation in H82 sgAPC cells as measured by AXIN2 mRNA levels by qPCR ( p

    Techniques Used: Construct, Activation Assay, Activity Assay, shRNA, Sequencing, Real-time Polymerase Chain Reaction, TOPFlash assay, Over Expression, CRISPR

    ASCL1 and MYCL are downregulated in post-chemotherapy human SCLC tissue and chemotherapy resistant cell lines. a Contingency table with number of chemotherapy naive (pre) and post-chemotherapy (post) SCLC human samples stained with antibodies to ASCL1. b Representative IHC for ASCL1 positive staining (brown) and negative staining (blue). Scale bar represents 20 μm. c RNA expression (counts) for indicated genes (ASCL1, MYCL) from matched pairs of chemotherapy-naive and resistant human SCLC cell lines performed in biological duplicate and compared using unpaired t- tests. H1048 NCI-H1048, P parental cells, CR cisplatin resistant, ECR etoposide and cisplatin resistant, * p
    Figure Legend Snippet: ASCL1 and MYCL are downregulated in post-chemotherapy human SCLC tissue and chemotherapy resistant cell lines. a Contingency table with number of chemotherapy naive (pre) and post-chemotherapy (post) SCLC human samples stained with antibodies to ASCL1. b Representative IHC for ASCL1 positive staining (brown) and negative staining (blue). Scale bar represents 20 μm. c RNA expression (counts) for indicated genes (ASCL1, MYCL) from matched pairs of chemotherapy-naive and resistant human SCLC cell lines performed in biological duplicate and compared using unpaired t- tests. H1048 NCI-H1048, P parental cells, CR cisplatin resistant, ECR etoposide and cisplatin resistant, * p

    Techniques Used: Staining, Immunohistochemistry, Negative Staining, RNA Expression

    6) Product Images from "Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53"

    Article Title: Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1317-7

    Crbn deficiency elevates the mortality rate of mice upon etoposide challenge. a Crbn deficiency promotes the death of mice after etoposide injection. Wild-type (WT) (n = 8) and Crbn knockout (KO) ( n = 8) mice were intraperitoneally injected with 100 mg/kg etoposide. Mice were monitored for 4 days. * P
    Figure Legend Snippet: Crbn deficiency elevates the mortality rate of mice upon etoposide challenge. a Crbn deficiency promotes the death of mice after etoposide injection. Wild-type (WT) (n = 8) and Crbn knockout (KO) ( n = 8) mice were intraperitoneally injected with 100 mg/kg etoposide. Mice were monitored for 4 days. * P

    Techniques Used: Mouse Assay, Injection, Knock-Out

    Cereblon (CRBN) deficiency decreases the viability of fibroblasts upon DNA damage. a CRBN depletion increases propidium iodide (PI)-positive primary fibroblasts upon etoposide or cisplatin treatment. Primary fibroblasts from wild-type (WT) and Crbn knockout (KO) littermate mice were exposed to etoposide (50 μM) or cisplatin (10 μM) for 48 h and then subjected to Hoechst and PI staining. Scale bar: 20 µm. b Quantitative data (mean ± SD) of a from three independent experiments. ** P
    Figure Legend Snippet: Cereblon (CRBN) deficiency decreases the viability of fibroblasts upon DNA damage. a CRBN depletion increases propidium iodide (PI)-positive primary fibroblasts upon etoposide or cisplatin treatment. Primary fibroblasts from wild-type (WT) and Crbn knockout (KO) littermate mice were exposed to etoposide (50 μM) or cisplatin (10 μM) for 48 h and then subjected to Hoechst and PI staining. Scale bar: 20 µm. b Quantitative data (mean ± SD) of a from three independent experiments. ** P

    Techniques Used: Knock-Out, Mouse Assay, Staining

    Cereblon (CRBN) inhibits etoposide-induced apoptosis in a p53-dependent manner. a Validation of knockdown efficiency for CRBN and p53 . Human embryonic kidney (HEK) 293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 48 h, lysed, and the resulting cell lysates were subjected to immunoblotting analysis using the indicated antibodies. b p53 knockdown attenuates the effect of CRBN on the mitochondrial membrane potential. HEK293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 40 h and then treated with etoposide (50 μM) for 8 h. Cells were stained with tetramethylrhodamine methyl ester (TMRM) and visualized under fluorescence microscope. Scale bar: 20 µm. c Quantitative data (mean ± SD) of b from three independent experiments. ** P
    Figure Legend Snippet: Cereblon (CRBN) inhibits etoposide-induced apoptosis in a p53-dependent manner. a Validation of knockdown efficiency for CRBN and p53 . Human embryonic kidney (HEK) 293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 48 h, lysed, and the resulting cell lysates were subjected to immunoblotting analysis using the indicated antibodies. b p53 knockdown attenuates the effect of CRBN on the mitochondrial membrane potential. HEK293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 40 h and then treated with etoposide (50 μM) for 8 h. Cells were stained with tetramethylrhodamine methyl ester (TMRM) and visualized under fluorescence microscope. Scale bar: 20 µm. c Quantitative data (mean ± SD) of b from three independent experiments. ** P

    Techniques Used: Transfection, Staining, Fluorescence, Microscopy

    7) Product Images from "Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies"

    Article Title: Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.7754

    Effects of etoposide on the DNA damage response pathways in KKU-M055 and KKU-M214 cells. The cells were treated with 4 Gy X-rays or etoposide alone (0.05 µg/ml) or pretreated with etoposide for 24 h prior to X-ray irradiation. The cells were collected at different time points for protein extraction and cell cycle analysis. (A) The levels of p53, p-p53 Ser15 and p21 in extracts of KKU-M055 and KKU-M214 cells, following etoposide treatment were determined by western blot analysis. (B) The levels of relevant proteins for G2 checkpoint signaling in extracts of KKU-M055 and KKU-M214 cells following etoposide treatment were determined by western blot analysis. The detection of actin was used as a loading control. (C) Cell cycle distribution profiles were analyzed by flow cytometry. The numbers in the histograms indicate the percentages of the cells in each phase of the cell cycle (G 1 , S and G 2 /M) or AP. IR, irradiation; p-p53 Ser15, tumor protein p53 phosphorylated at Ser15; p21, cyclin-dependent kinase inhibitor 1A; p-Chk Thr68, checkpoint kinase 2 phosphorylated at Thr68; Wee1, Wee1-like protein kinase; Cdc2, cyclin-dependent kinase 1; AP, aneuploidy.
    Figure Legend Snippet: Effects of etoposide on the DNA damage response pathways in KKU-M055 and KKU-M214 cells. The cells were treated with 4 Gy X-rays or etoposide alone (0.05 µg/ml) or pretreated with etoposide for 24 h prior to X-ray irradiation. The cells were collected at different time points for protein extraction and cell cycle analysis. (A) The levels of p53, p-p53 Ser15 and p21 in extracts of KKU-M055 and KKU-M214 cells, following etoposide treatment were determined by western blot analysis. (B) The levels of relevant proteins for G2 checkpoint signaling in extracts of KKU-M055 and KKU-M214 cells following etoposide treatment were determined by western blot analysis. The detection of actin was used as a loading control. (C) Cell cycle distribution profiles were analyzed by flow cytometry. The numbers in the histograms indicate the percentages of the cells in each phase of the cell cycle (G 1 , S and G 2 /M) or AP. IR, irradiation; p-p53 Ser15, tumor protein p53 phosphorylated at Ser15; p21, cyclin-dependent kinase inhibitor 1A; p-Chk Thr68, checkpoint kinase 2 phosphorylated at Thr68; Wee1, Wee1-like protein kinase; Cdc2, cyclin-dependent kinase 1; AP, aneuploidy.

    Techniques Used: Irradiation, Protein Extraction, Cell Cycle Assay, Western Blot, Flow Cytometry, Cytometry

    Distinct modes of cell death induced by etoposide or radiation or a combination thereof in KKU-M055 and KKU-M214 cells. The cells were pretreated with 0.05 µg/ml etoposide for 24 h. Subsequently, the cells were irradiated with X-rays (4 Gy) or left without irradiation. The control cells were neither treated with etoposide nor irradiated. After 24 and 48 h, the cells were stained with Hoechst 33342. Apoptosis, mitotic catastrophe or senescence was identified as described in the Materials and methods. (A) Representative images of nuclear staining with Hoechst 33342 indicates apoptotic (arrows), mitotic catastrophic (diamonds) and senescent cells (star). (B) The frequencies of apoptosis, mitotic catastrophe and senescence were quantified by fluorescence microscopy of Hoechst 33342 nuclear-stained cells. (C) Levels of key apoptotic proteins were determined by western blot analysis. The detection of actin was used as a loading control C, control cells; E, cells treated with etoposide; IR, irradiation; PARP, poly (ADP-ribose) polymerase; IR+ET, cells treated with a combination of X-rays and etoposide.
    Figure Legend Snippet: Distinct modes of cell death induced by etoposide or radiation or a combination thereof in KKU-M055 and KKU-M214 cells. The cells were pretreated with 0.05 µg/ml etoposide for 24 h. Subsequently, the cells were irradiated with X-rays (4 Gy) or left without irradiation. The control cells were neither treated with etoposide nor irradiated. After 24 and 48 h, the cells were stained with Hoechst 33342. Apoptosis, mitotic catastrophe or senescence was identified as described in the Materials and methods. (A) Representative images of nuclear staining with Hoechst 33342 indicates apoptotic (arrows), mitotic catastrophic (diamonds) and senescent cells (star). (B) The frequencies of apoptosis, mitotic catastrophe and senescence were quantified by fluorescence microscopy of Hoechst 33342 nuclear-stained cells. (C) Levels of key apoptotic proteins were determined by western blot analysis. The detection of actin was used as a loading control C, control cells; E, cells treated with etoposide; IR, irradiation; PARP, poly (ADP-ribose) polymerase; IR+ET, cells treated with a combination of X-rays and etoposide.

    Techniques Used: Irradiation, Staining, Fluorescence, Microscopy, Western Blot

    Effects of etoposide on the radiosensitivities of cholangiocarcinoma cell lines. The cell survival curves of (A) KKU-M055 and (B) KKU-M214 cells were obtained from clonogenic survival assays. The cells were treated with X-ray irradiation or etoposide (0.025 or 0.05 µg/ml) alone or pretreated with etoposide for 24 h prior to X-ray irradiation. Survival fractions were determined at day 10 following X-ray irradiation. The dose-response curves depict the mean ± standard deviation of survival fractions of three independent experiments. IR, irradiation.
    Figure Legend Snippet: Effects of etoposide on the radiosensitivities of cholangiocarcinoma cell lines. The cell survival curves of (A) KKU-M055 and (B) KKU-M214 cells were obtained from clonogenic survival assays. The cells were treated with X-ray irradiation or etoposide (0.025 or 0.05 µg/ml) alone or pretreated with etoposide for 24 h prior to X-ray irradiation. Survival fractions were determined at day 10 following X-ray irradiation. The dose-response curves depict the mean ± standard deviation of survival fractions of three independent experiments. IR, irradiation.

    Techniques Used: Irradiation, Standard Deviation

    8) Product Images from "Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease"

    Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddx046

    SK2 promotes formation of DNA DSBs in primary neurons. ( A ) Cortical neurons were transfected with mApple (control) or with SK2-mApple-NLS. At 24 h after transfection, cells were fixed, stained with an antibody against γH2A.X (a marker of DSBs) and with the nuclear Hoechst dye (DAPI), and imaged. As a positive control, neurons were treated with the DNA damaging drug etoposide. Note the γH2A.X puncta index in cells treated with etoposide and in SK2-mApple-NLS-expressing neurons is significantly greater than non-treated mApple-expressing neurons. Scale bar is 5 μm. See right panel for higher magnification image of γH2A.X. Scale bar is 2 μm. ( B ) Quantification of the γH2A.X puncta index in cortical and striatal neurons transfected with mApple (cont, control) or with SK2-mApple-NLS or transfected with mApple and treated with 5 µM etoposide (etop). *** P
    Figure Legend Snippet: SK2 promotes formation of DNA DSBs in primary neurons. ( A ) Cortical neurons were transfected with mApple (control) or with SK2-mApple-NLS. At 24 h after transfection, cells were fixed, stained with an antibody against γH2A.X (a marker of DSBs) and with the nuclear Hoechst dye (DAPI), and imaged. As a positive control, neurons were treated with the DNA damaging drug etoposide. Note the γH2A.X puncta index in cells treated with etoposide and in SK2-mApple-NLS-expressing neurons is significantly greater than non-treated mApple-expressing neurons. Scale bar is 5 μm. See right panel for higher magnification image of γH2A.X. Scale bar is 2 μm. ( B ) Quantification of the γH2A.X puncta index in cortical and striatal neurons transfected with mApple (cont, control) or with SK2-mApple-NLS or transfected with mApple and treated with 5 µM etoposide (etop). *** P

    Techniques Used: Transfection, Staining, Marker, Positive Control, Expressing

    9) Product Images from "An internal ribosome entry site in the coding region of tyrosyl-DNA phosphodiesterase 2 drives alternative translation start"

    Article Title: An internal ribosome entry site in the coding region of tyrosyl-DNA phosphodiesterase 2 drives alternative translation start

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006269

    ΔN-TDP2 is catalytically active. A, representative results of GST-tagged TDP2 incubated with T5PNP to assay phosphodiesterase catalytic activity. Data points are average ± S.D. of duplicates. B, immunoblot of TDP2 −/− ) reinfected with a retrovirus to re-express TDP2 with an IRES-GFP marker. Lysates are from the entire infected, GFP+ population. Constructs included FL- TDP2 , ΔN- TDP2 , mNLS- TDP2 , M54L- TDP2 , and two phosphodiesterase-dead mutants, E152A- TDP2 and D262A- TDP2. C, the reinfected cell lines were treated with the indicated concentration of etoposide for 72 h. % survival was determined by crystal violet staining and normalization to untreated cells. Data points are mean ± S.D. of experimental triplicates. D, close-up of boxed area in C visualizing absolute EC 50 values.
    Figure Legend Snippet: ΔN-TDP2 is catalytically active. A, representative results of GST-tagged TDP2 incubated with T5PNP to assay phosphodiesterase catalytic activity. Data points are average ± S.D. of duplicates. B, immunoblot of TDP2 −/− ) reinfected with a retrovirus to re-express TDP2 with an IRES-GFP marker. Lysates are from the entire infected, GFP+ population. Constructs included FL- TDP2 , ΔN- TDP2 , mNLS- TDP2 , M54L- TDP2 , and two phosphodiesterase-dead mutants, E152A- TDP2 and D262A- TDP2. C, the reinfected cell lines were treated with the indicated concentration of etoposide for 72 h. % survival was determined by crystal violet staining and normalization to untreated cells. Data points are mean ± S.D. of experimental triplicates. D, close-up of boxed area in C visualizing absolute EC 50 values.

    Techniques Used: Incubation, Activity Assay, Marker, Infection, Construct, Concentration Assay, Staining

    10) Product Images from "Histone deacetylase inhibitor sodium butyrate suppresses DNA double strand break repair induced by etoposide more effectively in MCF-7 cells than in HEK293 cells"

    Article Title: Histone deacetylase inhibitor sodium butyrate suppresses DNA double strand break repair induced by etoposide more effectively in MCF-7 cells than in HEK293 cells

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-014-0030-5

    Sodium butyrate enhances the cytotoxic effects of etoposide, and the effect is more dramatic in MCF-7 cells than in HEK293 cells. (A) MCF-7 and HEK293 cells were pre-treated with DMSO vehicle or 2 mM sodium butyrate for 4 h, and then were treated with vehicle control or 0.5 μM etoposide for 20 h. After the treatment period, cells were re-seeded for colony forming assay and grown for 15 days. The colonies were stained with 0.5% methylene blue for visualization. Results are representative of 3 independent experiments. (B) HEK293 and MCF-7 cells were treated for 4 h with 0, 0.5, 2.0, or 8.0 mM sodium butyrate and then were exposed to DMSO vehicle or 0.5 μM etoposide for 20 h. After the treatment period, cells were re-seeded for colony forming assay. The fraction of surviving colonies was determined for the 0.5, 2.0, and 8.0 mM sodium butyrate groups relative to the corresponding 0 mM sodium butyrate group.* P
    Figure Legend Snippet: Sodium butyrate enhances the cytotoxic effects of etoposide, and the effect is more dramatic in MCF-7 cells than in HEK293 cells. (A) MCF-7 and HEK293 cells were pre-treated with DMSO vehicle or 2 mM sodium butyrate for 4 h, and then were treated with vehicle control or 0.5 μM etoposide for 20 h. After the treatment period, cells were re-seeded for colony forming assay and grown for 15 days. The colonies were stained with 0.5% methylene blue for visualization. Results are representative of 3 independent experiments. (B) HEK293 and MCF-7 cells were treated for 4 h with 0, 0.5, 2.0, or 8.0 mM sodium butyrate and then were exposed to DMSO vehicle or 0.5 μM etoposide for 20 h. After the treatment period, cells were re-seeded for colony forming assay. The fraction of surviving colonies was determined for the 0.5, 2.0, and 8.0 mM sodium butyrate groups relative to the corresponding 0 mM sodium butyrate group.* P

    Techniques Used: Staining

    Sodium butyrate statistically increases γ-H2AX foci induced by etoposide in MCF-7 cells but not HEK293 cells. HEK293 and MCF-7 cells growing on slides in 24 well plates were exposed to DMSO vehicle or 2.0 mM sodium butyrate for 4 h followed by 20 h DMSO vehicle or 10 μM etoposide treatment. Following the treatment period, cells were fixed and assessed for γ-H2AX foci. (A) The average numbers of γ-H2AX foci per cell are shown. The inset shows a representative image of staining for a γ-H2AX-positive cell. (B) The % of foci-containing cells with > 5 foci was calculated. * P
    Figure Legend Snippet: Sodium butyrate statistically increases γ-H2AX foci induced by etoposide in MCF-7 cells but not HEK293 cells. HEK293 and MCF-7 cells growing on slides in 24 well plates were exposed to DMSO vehicle or 2.0 mM sodium butyrate for 4 h followed by 20 h DMSO vehicle or 10 μM etoposide treatment. Following the treatment period, cells were fixed and assessed for γ-H2AX foci. (A) The average numbers of γ-H2AX foci per cell are shown. The inset shows a representative image of staining for a γ-H2AX-positive cell. (B) The % of foci-containing cells with > 5 foci was calculated. * P

    Techniques Used: Staining

    Sodium butyrate modulates the nuclear expression of DSB repair proteins induced by etoposide in MCF-7 and MEL293 cells. HEK293 and MCF-7 cells were pre-treated with DMSO vehicle or 0.5, 2.0, 8.0 mM sodium butyrate for 4 h before exposure to DMSO vehicle or 10 μM etoposide for 20 h. After the treatment period, the cells were harvested, and then soluble nuclear protein and chromatin bound protein were extracted using a subcellular protein fraction kit. 50 μg proteins were loaded for western blot analysis. Coomassie blue staining gel is shown as loading control. Results are representative of three independent experiments.
    Figure Legend Snippet: Sodium butyrate modulates the nuclear expression of DSB repair proteins induced by etoposide in MCF-7 and MEL293 cells. HEK293 and MCF-7 cells were pre-treated with DMSO vehicle or 0.5, 2.0, 8.0 mM sodium butyrate for 4 h before exposure to DMSO vehicle or 10 μM etoposide for 20 h. After the treatment period, the cells were harvested, and then soluble nuclear protein and chromatin bound protein were extracted using a subcellular protein fraction kit. 50 μg proteins were loaded for western blot analysis. Coomassie blue staining gel is shown as loading control. Results are representative of three independent experiments.

    Techniques Used: Expressing, Western Blot, Staining

    11) Product Images from "Ezrin Enhances EGFR Signaling and Modulates Erlotinib Sensitivity in Non–Small Cell Lung Cancer Cells"

    Article Title: Ezrin Enhances EGFR Signaling and Modulates Erlotinib Sensitivity in Non–Small Cell Lung Cancer Cells

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

    doi: 10.1016/j.neo.2016.01.002

    Ezrin knockdown sensitizes NSCLC cells to erlotinib treatment. MTT test was used to evaluate the cell viability. (A) Nonlinear curve fit and (B) IC 50 values of control siRNA (siCTRL)– and siEZR-treated A549, H292, and H520 cells are given. Reduced ezrin expression caused a decreased IC 50 value for erlotinib in A549 and H292 cells compared with H520 cells. (C) Ezrin inhibitor NSC305787 shows synergism with erlotinib in A549 cells. No synergy was observed in H520 cells treated with erlotinib-NSC305787 combination (D) and in A549 cells treated with etoposide-NSC305787 combination (E).
    Figure Legend Snippet: Ezrin knockdown sensitizes NSCLC cells to erlotinib treatment. MTT test was used to evaluate the cell viability. (A) Nonlinear curve fit and (B) IC 50 values of control siRNA (siCTRL)– and siEZR-treated A549, H292, and H520 cells are given. Reduced ezrin expression caused a decreased IC 50 value for erlotinib in A549 and H292 cells compared with H520 cells. (C) Ezrin inhibitor NSC305787 shows synergism with erlotinib in A549 cells. No synergy was observed in H520 cells treated with erlotinib-NSC305787 combination (D) and in A549 cells treated with etoposide-NSC305787 combination (E).

    Techniques Used: MTT Assay, Expressing

    12) Product Images from "FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation"

    Article Title: FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation

    Journal: Oncogene

    doi: 10.1038/onc.2016.247

    Regorafenib-resistant cells are re-sensitized by Mcl-1 inhibition, and cross-resistant to other anticancer agents that induce Mcl-1 degradation (A) Western blotting of indicated proteins in parental and regorafenib-resistant HCT116, Lim2405 and SW480 cells treated with 40 μM regorafenib at indicated time points. p-ERK: Thr202/Tyr204; p-GSK3β: Ser9. (B) HCT116-R cells transfected with control or Mcl-1 siRNA were treated with 40 μM regorafenib for 48 hr. Left , western blot analysis of Mcl-1 knockdown; right , analysis of apoptosis by counting condensed and fragmented nuclei after nuclear staining. (C) HCT116-R cells were treated for 48 hr with 40 μM regorafenib alone or in combination with 1 μM of the Mcl-1 inhibitor TW-37 or the Bcl-2/Bcl-X L inhibitor ABT-737. Apoptosis was analyzed as in (B). (D) Parental and regorafenib-resistant HCT116 cells were treated with 40 μM regorafenib, 20 μM sorafenib, 1 μM UCN-01, 1 μM YM-155, 10 μM roscovitine, 15 μM sunitinib, 10 μM crizotinib, 10 nM TRAIL, 10 μM VX680, 20 μM etoposide, 20 μM temsirolimus, or 120 μM sulindac sulfide for 48 hr. Apoptosis was analyzed as in (B). (E) Western blotting of Mcl-1 in parental and regorafenib-resistant HCT116 cells treated with indicated agents as in (D) for 24 hr. Results in (B)-(D) represent the means ± s.d. of three independent experiments. NS, P > 0.05; *, P
    Figure Legend Snippet: Regorafenib-resistant cells are re-sensitized by Mcl-1 inhibition, and cross-resistant to other anticancer agents that induce Mcl-1 degradation (A) Western blotting of indicated proteins in parental and regorafenib-resistant HCT116, Lim2405 and SW480 cells treated with 40 μM regorafenib at indicated time points. p-ERK: Thr202/Tyr204; p-GSK3β: Ser9. (B) HCT116-R cells transfected with control or Mcl-1 siRNA were treated with 40 μM regorafenib for 48 hr. Left , western blot analysis of Mcl-1 knockdown; right , analysis of apoptosis by counting condensed and fragmented nuclei after nuclear staining. (C) HCT116-R cells were treated for 48 hr with 40 μM regorafenib alone or in combination with 1 μM of the Mcl-1 inhibitor TW-37 or the Bcl-2/Bcl-X L inhibitor ABT-737. Apoptosis was analyzed as in (B). (D) Parental and regorafenib-resistant HCT116 cells were treated with 40 μM regorafenib, 20 μM sorafenib, 1 μM UCN-01, 1 μM YM-155, 10 μM roscovitine, 15 μM sunitinib, 10 μM crizotinib, 10 nM TRAIL, 10 μM VX680, 20 μM etoposide, 20 μM temsirolimus, or 120 μM sulindac sulfide for 48 hr. Apoptosis was analyzed as in (B). (E) Western blotting of Mcl-1 in parental and regorafenib-resistant HCT116 cells treated with indicated agents as in (D) for 24 hr. Results in (B)-(D) represent the means ± s.d. of three independent experiments. NS, P > 0.05; *, P

    Techniques Used: Inhibition, Western Blot, Transfection, Staining

    13) Product Images from "The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation"

    Article Title: The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16394-2

    Induction of p53 and appearance of post-mitotic G1 (2N) cells by PLK1 inhibitors occurs through the DNA damage response pathways. ( A ) HCT116-p53+/+ cells were pre-treated for 1 h with 10 μM KU55933 and/or 10 μM VE821, and subsequently treated for 24 h with 20 nM GSK461364 or, as control, 50 μM etoposide. Cell extracts were analyzed by western blotting. ( B ) HCT116-p53+/+ and -p53−/− cells were pre-treated for 1 h with 10 μM KU and 10μM VE821 then further treated for 24 h with 20 nM GSK461364 or 10 nM BI6727, or with DMSO as control. Cells were then harvested and analyzed by flow cytometry. ( C ) H1299 (endogenous p53-null) cells ectopically expressing wild type p53, or a S15A substitution mutant of p53, via the LacSwitch II system (Stratagene) were treated for 16 h with increasing levels of the inducer, IPTG. Cell extracts were analyzed by western blotting as indicated. ( D ) H1299-WTp53 or H1299-S15A-p53 cells were treated for 16 h with 100 μM IPTG, followed by treatment for 24 h with 20 nM GSK461364 or 10 nM BI6727, or DMSO as control. Cells were harvested and analyzed by flow cytometry. Panels A and C show cropped western blots: full length gels including molecular weight markers are provided in the Supplementary Information. The data in panels B and D are each representative of two independent experiments, each done in triplicate. Error bars represent the standard deviation of the mean.
    Figure Legend Snippet: Induction of p53 and appearance of post-mitotic G1 (2N) cells by PLK1 inhibitors occurs through the DNA damage response pathways. ( A ) HCT116-p53+/+ cells were pre-treated for 1 h with 10 μM KU55933 and/or 10 μM VE821, and subsequently treated for 24 h with 20 nM GSK461364 or, as control, 50 μM etoposide. Cell extracts were analyzed by western blotting. ( B ) HCT116-p53+/+ and -p53−/− cells were pre-treated for 1 h with 10 μM KU and 10μM VE821 then further treated for 24 h with 20 nM GSK461364 or 10 nM BI6727, or with DMSO as control. Cells were then harvested and analyzed by flow cytometry. ( C ) H1299 (endogenous p53-null) cells ectopically expressing wild type p53, or a S15A substitution mutant of p53, via the LacSwitch II system (Stratagene) were treated for 16 h with increasing levels of the inducer, IPTG. Cell extracts were analyzed by western blotting as indicated. ( D ) H1299-WTp53 or H1299-S15A-p53 cells were treated for 16 h with 100 μM IPTG, followed by treatment for 24 h with 20 nM GSK461364 or 10 nM BI6727, or DMSO as control. Cells were harvested and analyzed by flow cytometry. Panels A and C show cropped western blots: full length gels including molecular weight markers are provided in the Supplementary Information. The data in panels B and D are each representative of two independent experiments, each done in triplicate. Error bars represent the standard deviation of the mean.

    Techniques Used: Western Blot, Flow Cytometry, Cytometry, Expressing, Mutagenesis, Molecular Weight, Standard Deviation

    14) Product Images from "Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer"

    Article Title: Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06162-9

    Loss of APC induces chemotherapy resistance in human SCLC cell lines. a Left: knockdown of APC in H1694 cells with two different shAPC constructs (shAPC#1 and shAPC#2); right: activation of WNT signaling as measured by AXIN2 upregulation and TOPFlash reporter activity (signal fold changes) in these cells. Control cells expressed shRNA with scrambled target sequence (shScr). APC and AXIN2 mRNA levels were measured by quantitative PCR (qPCR). Fold change is reported with respect to control cells, and values were compared using unpaired t tests. Each experiment was performed in biological triplicate (TOPFlash assay results for shAPC#2 are shown from n = 5 experiments). b Percentage of H1694 cells surviving etoposide following 72-h treatment. c Left: fold change in etoposide IC50 following APC knockdown in H1694 cells compared to control cells, and right: fold change in etoposide IC50 in APC knockdown (shAPC#2) cells following overexpression of APC or GFP (control). IC50 values were compared using ratio-paired t tests. d Results from Surveyor assay demonstrating genomic alterations in APC (cleavage products indicated by black bar) following CRISPR–Cas9-guided deletion in H82 sgAPC cells and in-frame deletions in APC that were identified through targeted sequencing of the APC sgRNA site. e WNT activation in H82 sgAPC cells as measured by AXIN2 mRNA levels by qPCR ( p
    Figure Legend Snippet: Loss of APC induces chemotherapy resistance in human SCLC cell lines. a Left: knockdown of APC in H1694 cells with two different shAPC constructs (shAPC#1 and shAPC#2); right: activation of WNT signaling as measured by AXIN2 upregulation and TOPFlash reporter activity (signal fold changes) in these cells. Control cells expressed shRNA with scrambled target sequence (shScr). APC and AXIN2 mRNA levels were measured by quantitative PCR (qPCR). Fold change is reported with respect to control cells, and values were compared using unpaired t tests. Each experiment was performed in biological triplicate (TOPFlash assay results for shAPC#2 are shown from n = 5 experiments). b Percentage of H1694 cells surviving etoposide following 72-h treatment. c Left: fold change in etoposide IC50 following APC knockdown in H1694 cells compared to control cells, and right: fold change in etoposide IC50 in APC knockdown (shAPC#2) cells following overexpression of APC or GFP (control). IC50 values were compared using ratio-paired t tests. d Results from Surveyor assay demonstrating genomic alterations in APC (cleavage products indicated by black bar) following CRISPR–Cas9-guided deletion in H82 sgAPC cells and in-frame deletions in APC that were identified through targeted sequencing of the APC sgRNA site. e WNT activation in H82 sgAPC cells as measured by AXIN2 mRNA levels by qPCR ( p

    Techniques Used: Construct, Activation Assay, Activity Assay, shRNA, Sequencing, Real-time Polymerase Chain Reaction, TOPFlash assay, Over Expression, CRISPR

    ASCL1 and MYCL are downregulated in post-chemotherapy human SCLC tissue and chemotherapy resistant cell lines. a Contingency table with number of chemotherapy naive (pre) and post-chemotherapy (post) SCLC human samples stained with antibodies to ASCL1. b Representative IHC for ASCL1 positive staining (brown) and negative staining (blue). Scale bar represents 20 μm. c RNA expression (counts) for indicated genes (ASCL1, MYCL) from matched pairs of chemotherapy-naive and resistant human SCLC cell lines performed in biological duplicate and compared using unpaired t- tests. H1048 NCI-H1048, P parental cells, CR cisplatin resistant, ECR etoposide and cisplatin resistant, * p
    Figure Legend Snippet: ASCL1 and MYCL are downregulated in post-chemotherapy human SCLC tissue and chemotherapy resistant cell lines. a Contingency table with number of chemotherapy naive (pre) and post-chemotherapy (post) SCLC human samples stained with antibodies to ASCL1. b Representative IHC for ASCL1 positive staining (brown) and negative staining (blue). Scale bar represents 20 μm. c RNA expression (counts) for indicated genes (ASCL1, MYCL) from matched pairs of chemotherapy-naive and resistant human SCLC cell lines performed in biological duplicate and compared using unpaired t- tests. H1048 NCI-H1048, P parental cells, CR cisplatin resistant, ECR etoposide and cisplatin resistant, * p

    Techniques Used: Staining, Immunohistochemistry, Negative Staining, RNA Expression

    15) Product Images from "Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis"

    Article Title: Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02950-5

    Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments
    Figure Legend Snippet: Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments

    Techniques Used: Irradiation, Immunoprecipitation, Sequencing, Concentration Assay, Purification, Knock-Out, Transfection, Activity Assay, FLAG-tag, Incubation, Recombinant, Acetylation Assay, Staining

    16) Product Images from "PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells"

    Article Title: PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells

    Journal: Oncogene

    doi: 10.1038/s41388-019-0725-6

    FOXM1 inhibition sensitizes PDAC cells to Etoposide and Gemcitabine A. PANC1 (left panel) and MIA PaCa2 (right panel) cells were treated with FDI6 20μM with or without Etoposide (Eto) 10μM for 48 hours (PANC1) or 24 hours (MIA PaCa2). The cells were lysed and cleaved PARP was analyzed by Western Blot. B. PANC1 (left panel) and MIA PaCa2 (right panel) cells were treated with FDI6 20μM with or without Gemcitabine (Gem) 10μM (PANC1) or 5μM (MIA PaCa2) for 48 hours or 24 hours respectively. The cells were lysed and the levels of cleaved PARP was analyzed by Western blot.
    Figure Legend Snippet: FOXM1 inhibition sensitizes PDAC cells to Etoposide and Gemcitabine A. PANC1 (left panel) and MIA PaCa2 (right panel) cells were treated with FDI6 20μM with or without Etoposide (Eto) 10μM for 48 hours (PANC1) or 24 hours (MIA PaCa2). The cells were lysed and cleaved PARP was analyzed by Western Blot. B. PANC1 (left panel) and MIA PaCa2 (right panel) cells were treated with FDI6 20μM with or without Gemcitabine (Gem) 10μM (PANC1) or 5μM (MIA PaCa2) for 48 hours or 24 hours respectively. The cells were lysed and the levels of cleaved PARP was analyzed by Western blot.

    Techniques Used: Inhibition, Western Blot

    PRRX1 isoforms limit DNA damage induced by IR, Etoposide and Gemcitabine PANC1 cells were transiently transfected with the pcDNA3.1(+), pcDNA3.1-myc-PRRX1A or pcDNA3.1-myc-PRRX1B plasmids. A. Then 48 hours post-transfection, the cells were irradiated with 0 or 8Gy and lysed 2 hours following irradiation. The levels of the DNA damage marker, phosphorylated H2Ax (Ser139), was analyzed by Western Blot. B. At 24 hours following transfection, the cells were treated with Etoposide 10μM or DMSO for 24 hours. The levels of cleaved PARP and phosphorylated H2Ax (Ser139), respectively a marker of apoptosis and DNA damage, were analyzed by Western Blot. C. At 24 hours following transfection, the cells were treated with Gemcitabine 10μM or DMSO for 24 hours. The cleaved PARP and phospho-H2Ax (Ser139) were analyzed by Western Blot.
    Figure Legend Snippet: PRRX1 isoforms limit DNA damage induced by IR, Etoposide and Gemcitabine PANC1 cells were transiently transfected with the pcDNA3.1(+), pcDNA3.1-myc-PRRX1A or pcDNA3.1-myc-PRRX1B plasmids. A. Then 48 hours post-transfection, the cells were irradiated with 0 or 8Gy and lysed 2 hours following irradiation. The levels of the DNA damage marker, phosphorylated H2Ax (Ser139), was analyzed by Western Blot. B. At 24 hours following transfection, the cells were treated with Etoposide 10μM or DMSO for 24 hours. The levels of cleaved PARP and phosphorylated H2Ax (Ser139), respectively a marker of apoptosis and DNA damage, were analyzed by Western Blot. C. At 24 hours following transfection, the cells were treated with Gemcitabine 10μM or DMSO for 24 hours. The cleaved PARP and phospho-H2Ax (Ser139) were analyzed by Western Blot.

    Techniques Used: Transfection, Irradiation, Marker, Western Blot

    17) Product Images from "Targeted Clinical Nanoparticles for Precision Cancer Therapy based on Disease-specific Molecular Inflection Points"

    Article Title: Targeted Clinical Nanoparticles for Precision Cancer Therapy based on Disease-specific Molecular Inflection Points

    Journal: Nano letters

    doi: 10.1021/acs.nanolett.7b04209

    Improved drug delivery with drug-carrying TNP. (A) Delivery of therapy with TNP caused cell death in the PSMA-expressing LNCaP cells (DoxoTNP: doxorubicin-loaded TNP). (B–C) Selective drug delivery of etoposide, paclitaxel and doxorubicin with TNP (LNCaP: FOLH1-positive cells; PC3-Ctrl: FOLH1-negative cells; *** P
    Figure Legend Snippet: Improved drug delivery with drug-carrying TNP. (A) Delivery of therapy with TNP caused cell death in the PSMA-expressing LNCaP cells (DoxoTNP: doxorubicin-loaded TNP). (B–C) Selective drug delivery of etoposide, paclitaxel and doxorubicin with TNP (LNCaP: FOLH1-positive cells; PC3-Ctrl: FOLH1-negative cells; *** P

    Techniques Used: Expressing

    18) Product Images from "Inhibition of the replication stress response is a synthetic vulnerability in SCLC that acts synergistically in combination with cisplatin"

    Article Title: Inhibition of the replication stress response is a synthetic vulnerability in SCLC that acts synergistically in combination with cisplatin

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-18-0972

    ATR inhibition in combination with cisplatin in vivo. A. Graph representing the average tumor volumes +/- standard error of the mean (SEM) of xenografted DMS-273 cells. B. Survival curve of mice bearing DMS-273 xenografts treated with the indicated compounds. End point of this analysis is the time until the xenografts reach a tumor volume of 1500 mm 3 . Average survival: Vehicle 13.4 ± 3.9; VE-822 12.5 ± 1.7; Cisplatin 15.3 ± 2.3; Cisplatin + Etoposide 18.3 ± 2.7 and Cisplatin + VE-822 26 ± 6.5 days. *p=0.002, **p=1.8x10 -5 C. Average tumor volumes +/- SEM of xenografted NCI-H187 cells. D. Survival curve of mice bearing NCI-H187 xenografts treated with the indicated compounds. End point of this analysis is the time until the xenografts reach a tumor volume of 1500 mm 3 . For all groups 12 mice were used. Average survival: Vehicle 25.5 ± 6.9; VE-822 27.8 ± 5.4; Cisplatin 27.4 ± 4.4; Cisplatin + Etoposide 27.1 ± 4.9 and Cisplatin + VE-822 39.2 ± 12 days. ***p=0.005
    Figure Legend Snippet: ATR inhibition in combination with cisplatin in vivo. A. Graph representing the average tumor volumes +/- standard error of the mean (SEM) of xenografted DMS-273 cells. B. Survival curve of mice bearing DMS-273 xenografts treated with the indicated compounds. End point of this analysis is the time until the xenografts reach a tumor volume of 1500 mm 3 . Average survival: Vehicle 13.4 ± 3.9; VE-822 12.5 ± 1.7; Cisplatin 15.3 ± 2.3; Cisplatin + Etoposide 18.3 ± 2.7 and Cisplatin + VE-822 26 ± 6.5 days. *p=0.002, **p=1.8x10 -5 C. Average tumor volumes +/- SEM of xenografted NCI-H187 cells. D. Survival curve of mice bearing NCI-H187 xenografts treated with the indicated compounds. End point of this analysis is the time until the xenografts reach a tumor volume of 1500 mm 3 . For all groups 12 mice were used. Average survival: Vehicle 25.5 ± 6.9; VE-822 27.8 ± 5.4; Cisplatin 27.4 ± 4.4; Cisplatin + Etoposide 27.1 ± 4.9 and Cisplatin + VE-822 39.2 ± 12 days. ***p=0.005

    Techniques Used: Inhibition, In Vivo, Mouse Assay

    Replication stress inhibition synergizes with DNA damaging agents in mouse cells. A. Graphs showing hypothetical combinations of two drugs in a combination matrix. The different doses of the drugs are indicated on the x- and y-axis. Graphs were produced using MacSynergyII software, which determines the degree of interaction between two drugs. Three potential outcomes are shown, with additive effects represented as a flat plane (left panel), mild synergism as a peak (middle panel)_and high synergy as a peak with an increase in volume. B . Graph showing the synergy score of the combination matrix of cisplatin and etoposide in a mSCLC cell line. C and D Synergy scores of the combinations of AZD7762 (CHK1 inhibitor) and VE-822 (ATR inhibitor) with etoposide (B) or cisplatin (C) in mSCLC. E. Synergy scores of the combinations of AZD7762 and VE-822 with cisplatin in primary MEFs. All synergy scores were calculated using the MAC Synergy II algorithm using data from three independent experiments.
    Figure Legend Snippet: Replication stress inhibition synergizes with DNA damaging agents in mouse cells. A. Graphs showing hypothetical combinations of two drugs in a combination matrix. The different doses of the drugs are indicated on the x- and y-axis. Graphs were produced using MacSynergyII software, which determines the degree of interaction between two drugs. Three potential outcomes are shown, with additive effects represented as a flat plane (left panel), mild synergism as a peak (middle panel)_and high synergy as a peak with an increase in volume. B . Graph showing the synergy score of the combination matrix of cisplatin and etoposide in a mSCLC cell line. C and D Synergy scores of the combinations of AZD7762 (CHK1 inhibitor) and VE-822 (ATR inhibitor) with etoposide (B) or cisplatin (C) in mSCLC. E. Synergy scores of the combinations of AZD7762 and VE-822 with cisplatin in primary MEFs. All synergy scores were calculated using the MAC Synergy II algorithm using data from three independent experiments.

    Techniques Used: Inhibition, Produced, Software

    19) Product Images from "Rapidly induced drug adaptation mediates escape from BRAF inhibition in single melanoma cells"

    Article Title: Rapidly induced drug adaptation mediates escape from BRAF inhibition in single melanoma cells

    Journal: bioRxiv

    doi: 10.1101/2020.03.15.992982

    Dabrafenib treatment induces quiescence incompletely. (A) A375 (left) and WM278 (right) cells were treated with 10 μM dabrafenib for the indicated durations and the levels of phospho-ERK and phospho-Rb S807/811 were measured by western blot (top). Signals were quantified by normalizing first to GAPDH levels then to the untreated condition (bottom). (B) Quantification of percentage of pRb + cells in SKMEL19 cells treated for indicated lengths of time with 0.1 μM, 1 μM, and 10 μM dabrafenib. The percentage of pRb + cells with 10 μM dabrafenib at 96 hr is noted. Error bars: as mean ± std of 3 replicate wells. (C) SKMEL19, A375 and WM278 dose-response curves showing the percent pRb + cells after 96 hr of dabrafenib treatment, determined by immunofluorescence quantification. For A375, the untreated 96 hr DMSO line falls at 60% (compared with 95% reported elsewhere in this study when cells were plated 24 hr before fixation) because 96 hr of unfettered growth on the plate results in partial contact inhibition. Error bars: as mean ± std of 3 replicate wells. (D) Apoptotic cell quantification by flow cytometric analyses of Annexin V-FITC and propidium iodide staining. DMSO bivariate plot is shown as a negative control for apoptosis; etoposide is shown as a positive control. Representative bivariate plot is shown for 96 hr of 1 μM dabrafenib. Quantified replicates of late apoptotic cells (Q2) in each treatment condition are shown for the indicated time points, with mean represented as a horizontal line. Error bars: as mean ± std of at least 3 replicate samples.
    Figure Legend Snippet: Dabrafenib treatment induces quiescence incompletely. (A) A375 (left) and WM278 (right) cells were treated with 10 μM dabrafenib for the indicated durations and the levels of phospho-ERK and phospho-Rb S807/811 were measured by western blot (top). Signals were quantified by normalizing first to GAPDH levels then to the untreated condition (bottom). (B) Quantification of percentage of pRb + cells in SKMEL19 cells treated for indicated lengths of time with 0.1 μM, 1 μM, and 10 μM dabrafenib. The percentage of pRb + cells with 10 μM dabrafenib at 96 hr is noted. Error bars: as mean ± std of 3 replicate wells. (C) SKMEL19, A375 and WM278 dose-response curves showing the percent pRb + cells after 96 hr of dabrafenib treatment, determined by immunofluorescence quantification. For A375, the untreated 96 hr DMSO line falls at 60% (compared with 95% reported elsewhere in this study when cells were plated 24 hr before fixation) because 96 hr of unfettered growth on the plate results in partial contact inhibition. Error bars: as mean ± std of 3 replicate wells. (D) Apoptotic cell quantification by flow cytometric analyses of Annexin V-FITC and propidium iodide staining. DMSO bivariate plot is shown as a negative control for apoptosis; etoposide is shown as a positive control. Representative bivariate plot is shown for 96 hr of 1 μM dabrafenib. Quantified replicates of late apoptotic cells (Q2) in each treatment condition are shown for the indicated time points, with mean represented as a horizontal line. Error bars: as mean ± std of at least 3 replicate samples.

    Techniques Used: Western Blot, Immunofluorescence, Inhibition, Staining, Negative Control, Positive Control

    20) Product Images from "Activation of PPARγ/P53 signaling is required for curcumin to induce hepatic stellate cell senescence"

    Article Title: Activation of PPARγ/P53 signaling is required for curcumin to induce hepatic stellate cell senescence

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.92

    Curcumin promoted senescence of activated HSC in vitro . HSCs were treated with DMSO (0.02%, w/v), etoposide or curcumin at indicated concentrations for 24 h. ( a ) Cell Count Kit-8 analysis of the ability of cell proliferation. * P
    Figure Legend Snippet: Curcumin promoted senescence of activated HSC in vitro . HSCs were treated with DMSO (0.02%, w/v), etoposide or curcumin at indicated concentrations for 24 h. ( a ) Cell Count Kit-8 analysis of the ability of cell proliferation. * P

    Techniques Used: In Vitro, Cell Counting

    21) Product Images from "XPO1 Inhibition Using Selinexor Synergizes With Chemotherapy in Acute Myeloid Leukemia (AML) by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus"

    Article Title: XPO1 Inhibition Using Selinexor Synergizes With Chemotherapy in Acute Myeloid Leukemia (AML) by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-15-2885

    Combination of selinexor and idarubicin or Topo IIα inhibitors results in synergistic inhibition of proliferation and induction of apoptosis in AML cells in vitro (A) Combination index (CI) plots of selinexor with idarubicin (IDA) and daunorubicin (DAUNO) concomitant treatment in AML cell lines MV4-11 and MOLM-13 and patient blasts (B) . The effect of the combinations was assessed by WST-1 assay after 48 hrs of concomitant drug treatment. The doses for both drugs were chosen according to their individual IC 50 ). (C) CI plots of selinexor with Topo IIα inhibitors, etoposide and mitoxantrone in MV4-11 and MOLM-13 AML cell lines. The effects of the combinations were calculated using CalcuSyn software, where CI
    Figure Legend Snippet: Combination of selinexor and idarubicin or Topo IIα inhibitors results in synergistic inhibition of proliferation and induction of apoptosis in AML cells in vitro (A) Combination index (CI) plots of selinexor with idarubicin (IDA) and daunorubicin (DAUNO) concomitant treatment in AML cell lines MV4-11 and MOLM-13 and patient blasts (B) . The effect of the combinations was assessed by WST-1 assay after 48 hrs of concomitant drug treatment. The doses for both drugs were chosen according to their individual IC 50 ). (C) CI plots of selinexor with Topo IIα inhibitors, etoposide and mitoxantrone in MV4-11 and MOLM-13 AML cell lines. The effects of the combinations were calculated using CalcuSyn software, where CI

    Techniques Used: Inhibition, In Vitro, WST-1 Assay, Software

    22) Product Images from "Etoposide Induces Mitochondrial Dysfunction and Cellular Senescence in Primary Cultured Rat Astrocytes"

    Article Title: Etoposide Induces Mitochondrial Dysfunction and Cellular Senescence in Primary Cultured Rat Astrocytes

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2019.151

    Functional changes of mitochondria in etoposide-treated astrocytes. Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 24 h. (A) Cultured astrocytes were detected for mitochondrial membrane potential by fluorescence staining of TMRM. Representative images were visualized by the IncuCyte ZOOM microscope software 2015A (Essen Bioscience) under the 20× object. The data were calculated by IncuCyte software (Essen Bioscience). (B) The representative graph indicates the total TMRM object integrated intensity (RCU × μm 2 /image). (C) The graph shows the total TMRM object area (μm 2 /image) in astrocytes. Bars represent the mean ± SEM. ** indicates p
    Figure Legend Snippet: Functional changes of mitochondria in etoposide-treated astrocytes. Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 24 h. (A) Cultured astrocytes were detected for mitochondrial membrane potential by fluorescence staining of TMRM. Representative images were visualized by the IncuCyte ZOOM microscope software 2015A (Essen Bioscience) under the 20× object. The data were calculated by IncuCyte software (Essen Bioscience). (B) The representative graph indicates the total TMRM object integrated intensity (RCU × μm 2 /image). (C) The graph shows the total TMRM object area (μm 2 /image) in astrocytes. Bars represent the mean ± SEM. ** indicates p

    Techniques Used: Functional Assay, Cell Culture, Fluorescence, Staining, Microscopy, Software

    Morphological changes of etoposide-treated astrocytes in vitro . (A) Astrocytes were stained to determine the activity of senescence-associated beta-galactosidase (SA-β-gal). Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 24 h. Stained astrocytes were taken at 100x magnification and black triangles indicate SA-β-gal-positive cells. The graph represents the fold change in SA-β-gal-positive cell number in 10 μM etoposidetreated astrocytes. (B) Astrocytes were stained to determine the nuclear size. The graph presents the percent of nuclear size compared to control. The bars represent the mean ± SEM. * indicates p
    Figure Legend Snippet: Morphological changes of etoposide-treated astrocytes in vitro . (A) Astrocytes were stained to determine the activity of senescence-associated beta-galactosidase (SA-β-gal). Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 24 h. Stained astrocytes were taken at 100x magnification and black triangles indicate SA-β-gal-positive cells. The graph represents the fold change in SA-β-gal-positive cell number in 10 μM etoposidetreated astrocytes. (B) Astrocytes were stained to determine the nuclear size. The graph presents the percent of nuclear size compared to control. The bars represent the mean ± SEM. * indicates p

    Techniques Used: In Vitro, Staining, Activity Assay

    Expression of inflammatory and cell cycle markers in etoposide-treated astrocytes in vitro . Rat primary cultured astrocytes were treated with DMSO (vehicle), etoposide 1 μM, or etoposide 10 μM for 24 h. (A) The expression level of senescence-related genes was measured by reverse transcription PCR (RT-PCR). The graph represents the mRNA expression levels. Values were normalized based on GAPDH. (B) The expression level of senescence-related proteins was measured by Western blot. Densitometry analysis of the protein expression levels. The graph represents the protein expression levels and the values were normalized to the Actin expression. The bars represent the mean ± SEM. * indicates p
    Figure Legend Snippet: Expression of inflammatory and cell cycle markers in etoposide-treated astrocytes in vitro . Rat primary cultured astrocytes were treated with DMSO (vehicle), etoposide 1 μM, or etoposide 10 μM for 24 h. (A) The expression level of senescence-related genes was measured by reverse transcription PCR (RT-PCR). The graph represents the mRNA expression levels. Values were normalized based on GAPDH. (B) The expression level of senescence-related proteins was measured by Western blot. Densitometry analysis of the protein expression levels. The graph represents the protein expression levels and the values were normalized to the Actin expression. The bars represent the mean ± SEM. * indicates p

    Techniques Used: Expressing, In Vitro, Cell Culture, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Cellular functional changes of etoposide-treated astrocytes in vitro . Scratch wound assay (A–D). The monolayer of rat primary cultured astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 72 h. (A) Representative images of astrocytes in the wound-healing assay. Blue lines indicate the initial scratch-wound area. Red lines show wound closure at each time-point. Images and data obtained using IncuCyte (Essen Bioscience) under the 10× object. (B) Cell migration every 3 h into the wound area, represented by wound width (μm) in the modified scratch-wound assay analysis. (C) The percentage of relative wound density in the scratch wound area was determined. (D) Etoposide-treated cells were assessed for its phagocytosis ability using E. coli BioParticles. Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 12 h. (E) The graph shows total red object area per well (μm 2 /well) every 10 min for 12 h. (F) The representative graph shows the total red object integrated intensity (RCU × μm 2 /image) every 10 min for 12 h. Lines represent the mean ± SEM. * indicates p
    Figure Legend Snippet: Cellular functional changes of etoposide-treated astrocytes in vitro . Scratch wound assay (A–D). The monolayer of rat primary cultured astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 72 h. (A) Representative images of astrocytes in the wound-healing assay. Blue lines indicate the initial scratch-wound area. Red lines show wound closure at each time-point. Images and data obtained using IncuCyte (Essen Bioscience) under the 10× object. (B) Cell migration every 3 h into the wound area, represented by wound width (μm) in the modified scratch-wound assay analysis. (C) The percentage of relative wound density in the scratch wound area was determined. (D) Etoposide-treated cells were assessed for its phagocytosis ability using E. coli BioParticles. Astrocytes were treated with DMSO (vehicle) or 10 μM etoposide for 12 h. (E) The graph shows total red object area per well (μm 2 /well) every 10 min for 12 h. (F) The representative graph shows the total red object integrated intensity (RCU × μm 2 /image) every 10 min for 12 h. Lines represent the mean ± SEM. * indicates p

    Techniques Used: Functional Assay, In Vitro, Scratch Wound Assay Assay, Cell Culture, Wound Healing Assay, Migration, Modification

    23) Product Images from "DT2216—a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas"

    Article Title: DT2216—a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-020-00928-9

    DT2216 can synergistically kill TCL PDX cells in combination with ABT199 in vitro . a The viability of DFTL-28776 PDX cells was determined 24 h after doxorubicin (Dox), etoposide (Eto), or vincristine (Vin) treatment. EC 50 , half maximal effective concentration. The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). b The viability of DFTL-28776 PDX cells was determined 24 h after ABT263, ABT199, A-1155463, or S63845 treatment. The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). c Bcl-xL, Bcl-2, Mcl-1, and VHL expressions in MyLa cells, DFTL-28776 PDX cells, and PLT. d DT2216 degraded Bcl-xL in DFTL-28776 PDX cells after treatment with indicated concentrations of DT2216 for 16 h. e VHL ligand (VHL-L) pretreatment blocked the degradation of Bcl-xL induced by DT2216 in DFTL-28776 PDX cells. The PDX cells were pretreated with 10 μM VHL-L for 2 h, and then treated with indicated concentrations of DT2216 for 16 h. f Proteasome inhibition with MG132 blocked the degradation of Bcl-xL induced by DT2216 in DFTL-28776 PDX cells. The PDX cells were pretreated with 1 μM MG-132 for 2 h and then treated with the indicated concentrations of DT2216 for 16 h. g The viability of DFTL-28776 PDX cells was determined 24 h after treatment with DT2216, ABT199, or DT2216 plus ABT199 (DT+199 at a ratio of 1:1). The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). h The combination index (CI) values of DT+199 group at EC 25 , EC 50 , and EC 75 , values calculated from the data presented in g are presented. i Combination of DT2216 with ABT199 induced apoptosis in PDX cells. Apoptosis was assayed after treatment with 0.1 μM DT2216, 0.1 μM ABT199, or combination of 0.1 μM DT2216 with 0.1 μM ABT199 for 24 h. PI, propidium iodide
    Figure Legend Snippet: DT2216 can synergistically kill TCL PDX cells in combination with ABT199 in vitro . a The viability of DFTL-28776 PDX cells was determined 24 h after doxorubicin (Dox), etoposide (Eto), or vincristine (Vin) treatment. EC 50 , half maximal effective concentration. The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). b The viability of DFTL-28776 PDX cells was determined 24 h after ABT263, ABT199, A-1155463, or S63845 treatment. The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). c Bcl-xL, Bcl-2, Mcl-1, and VHL expressions in MyLa cells, DFTL-28776 PDX cells, and PLT. d DT2216 degraded Bcl-xL in DFTL-28776 PDX cells after treatment with indicated concentrations of DT2216 for 16 h. e VHL ligand (VHL-L) pretreatment blocked the degradation of Bcl-xL induced by DT2216 in DFTL-28776 PDX cells. The PDX cells were pretreated with 10 μM VHL-L for 2 h, and then treated with indicated concentrations of DT2216 for 16 h. f Proteasome inhibition with MG132 blocked the degradation of Bcl-xL induced by DT2216 in DFTL-28776 PDX cells. The PDX cells were pretreated with 1 μM MG-132 for 2 h and then treated with the indicated concentrations of DT2216 for 16 h. g The viability of DFTL-28776 PDX cells was determined 24 h after treatment with DT2216, ABT199, or DT2216 plus ABT199 (DT+199 at a ratio of 1:1). The data presented are mean ± SD ( n = 2 independent assays, with 3 replicates in each assay). h The combination index (CI) values of DT+199 group at EC 25 , EC 50 , and EC 75 , values calculated from the data presented in g are presented. i Combination of DT2216 with ABT199 induced apoptosis in PDX cells. Apoptosis was assayed after treatment with 0.1 μM DT2216, 0.1 μM ABT199, or combination of 0.1 μM DT2216 with 0.1 μM ABT199 for 24 h. PI, propidium iodide

    Techniques Used: In Vitro, Concentration Assay, Inhibition

    24) Product Images from "Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53"

    Article Title: Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1317-7

    Crbn deficiency elevates the mortality rate of mice upon etoposide challenge. a Crbn deficiency promotes the death of mice after etoposide injection. Wild-type (WT) (n = 8) and Crbn knockout (KO) ( n = 8) mice were intraperitoneally injected with 100 mg/kg etoposide. Mice were monitored for 4 days. * P
    Figure Legend Snippet: Crbn deficiency elevates the mortality rate of mice upon etoposide challenge. a Crbn deficiency promotes the death of mice after etoposide injection. Wild-type (WT) (n = 8) and Crbn knockout (KO) ( n = 8) mice were intraperitoneally injected with 100 mg/kg etoposide. Mice were monitored for 4 days. * P

    Techniques Used: Mouse Assay, Injection, Knock-Out

    Cereblon (CRBN) deficiency decreases the viability of fibroblasts upon DNA damage. a CRBN depletion increases propidium iodide (PI)-positive primary fibroblasts upon etoposide or cisplatin treatment. Primary fibroblasts from wild-type (WT) and Crbn knockout (KO) littermate mice were exposed to etoposide (50 μM) or cisplatin (10 μM) for 48 h and then subjected to Hoechst and PI staining. Scale bar: 20 µm. b Quantitative data (mean ± SD) of a from three independent experiments. ** P
    Figure Legend Snippet: Cereblon (CRBN) deficiency decreases the viability of fibroblasts upon DNA damage. a CRBN depletion increases propidium iodide (PI)-positive primary fibroblasts upon etoposide or cisplatin treatment. Primary fibroblasts from wild-type (WT) and Crbn knockout (KO) littermate mice were exposed to etoposide (50 μM) or cisplatin (10 μM) for 48 h and then subjected to Hoechst and PI staining. Scale bar: 20 µm. b Quantitative data (mean ± SD) of a from three independent experiments. ** P

    Techniques Used: Knock-Out, Mouse Assay, Staining

    Cereblon (CRBN) inhibits etoposide-induced apoptosis in a p53-dependent manner. a Validation of knockdown efficiency for CRBN and p53 . Human embryonic kidney (HEK) 293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 48 h, lysed, and the resulting cell lysates were subjected to immunoblotting analysis using the indicated antibodies. b p53 knockdown attenuates the effect of CRBN on the mitochondrial membrane potential. HEK293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 40 h and then treated with etoposide (50 μM) for 8 h. Cells were stained with tetramethylrhodamine methyl ester (TMRM) and visualized under fluorescence microscope. Scale bar: 20 µm. c Quantitative data (mean ± SD) of b from three independent experiments. ** P
    Figure Legend Snippet: Cereblon (CRBN) inhibits etoposide-induced apoptosis in a p53-dependent manner. a Validation of knockdown efficiency for CRBN and p53 . Human embryonic kidney (HEK) 293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 48 h, lysed, and the resulting cell lysates were subjected to immunoblotting analysis using the indicated antibodies. b p53 knockdown attenuates the effect of CRBN on the mitochondrial membrane potential. HEK293 cells were transfected with si NC or si CRBN along with si NC or si p53 for 40 h and then treated with etoposide (50 μM) for 8 h. Cells were stained with tetramethylrhodamine methyl ester (TMRM) and visualized under fluorescence microscope. Scale bar: 20 µm. c Quantitative data (mean ± SD) of b from three independent experiments. ** P

    Techniques Used: Transfection, Staining, Fluorescence, Microscopy

    25) Product Images from "PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth"

    Article Title: PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13608-1

    K382Ac recognition by PBRM1 is critical for p53 function in ccRCC cells. a , b ACHN ( a ) or Caki-1 ( b ) control and PBRM1 shRNA knockdown cells were treated with 50 μM etoposide for the indicated time points. Lysates were analyzed by immunoblots. c RCC4 cells with or without p53 KO were transfected with vector or Flag-PBRM1 at the indicated amounts. Lysates were analyzed by immunoblots. d ACHN control and PBRM1 shRNA #1 knockdown cells were treated with 10 μM Nutlin-3a for the indicated time points. Lysates were analyzed by immunoblots. e RCC4 cells stably expressing GFP, wild-type PBRM1, or BD4* mutant PBRM1 were treated with 100 μM etoposide for the indicated time points. Lysates were analyzed by immunoblots. f RCC4 cells stably expressing GFP, wild-type or indicated PBRM1 mutants were treated with 100 μM etoposide for the indicated times, and lysates were examined via immunoblots. g , h RCC4 cells stably expressing GFP, wild-type or BD4* mutant PBRM1 were treated with 100 μM etoposide for 24 h. Cells were harvested for RT-qPCR ( g ) or ChIP followed by qPCR ( h ). Data are shown as mean ± SEM. p-values were calculated using the paired two-tailed Student’s t -test. i , j ACHN control and PBRM1 shRNA knockdown cells were treated with vehicle or 50 μM etoposide for cell cycle analysis. Representative data is shown in i . Quantification of cell cycle phase is shown in j . Source data are provided as a Source Data file.
    Figure Legend Snippet: K382Ac recognition by PBRM1 is critical for p53 function in ccRCC cells. a , b ACHN ( a ) or Caki-1 ( b ) control and PBRM1 shRNA knockdown cells were treated with 50 μM etoposide for the indicated time points. Lysates were analyzed by immunoblots. c RCC4 cells with or without p53 KO were transfected with vector or Flag-PBRM1 at the indicated amounts. Lysates were analyzed by immunoblots. d ACHN control and PBRM1 shRNA #1 knockdown cells were treated with 10 μM Nutlin-3a for the indicated time points. Lysates were analyzed by immunoblots. e RCC4 cells stably expressing GFP, wild-type PBRM1, or BD4* mutant PBRM1 were treated with 100 μM etoposide for the indicated time points. Lysates were analyzed by immunoblots. f RCC4 cells stably expressing GFP, wild-type or indicated PBRM1 mutants were treated with 100 μM etoposide for the indicated times, and lysates were examined via immunoblots. g , h RCC4 cells stably expressing GFP, wild-type or BD4* mutant PBRM1 were treated with 100 μM etoposide for 24 h. Cells were harvested for RT-qPCR ( g ) or ChIP followed by qPCR ( h ). Data are shown as mean ± SEM. p-values were calculated using the paired two-tailed Student’s t -test. i , j ACHN control and PBRM1 shRNA knockdown cells were treated with vehicle or 50 μM etoposide for cell cycle analysis. Representative data is shown in i . Quantification of cell cycle phase is shown in j . Source data are provided as a Source Data file.

    Techniques Used: shRNA, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test, Cell Cycle Assay

    Mutation of BD4 abolishes the tumor suppressive function of PBRM1. a , b GFP, wild-type or BD4* mutant PBRM1 were stably expressed in Ren-01 PBRM1 KO cells (combination of three clones) ( a ) or PBRM1-null SLR24 cells ( b ). Cells were treated with 50 μM etoposide for the indicated times, and lysates were analyzed via immunoblots with indicated antibodies. The band intensity of indicated protein was measured with Bio-rad Image Lab 4.1, and the relative ratios were calculated over the signal intensity of Vinculin in the corresponding lanes. c – e . Nude mice xenograft analyses were performed with Ren-01 PBRM1 KO cells expressing GFP (left flank) or PBRM1 (right flank). Representative photographs of a mouse ( c , top) and tumors ( c , bottom). Tumors were excised and weighed, and data are presented as mean ± SEM ( d ). p-values were calculated using the paired two-tailed Student’s t -test. Tumors were stained for PBRM1 and p21 expression via IHC ( e ). Scale bar: 200 μm. f – h Nude mice xenograft analyses were performed with Ren-01 PBRM1 KO cells expressing wild-type PBRM1 (left flank) or the BD4* mutant PBRM1 (right flank). Tumors were excised and weighed, and data are presented as mean ± SEM ( g ). p -values were calculated using the paired two-tailed Student’s t -test. Representative photographs of a mouse ( f , top) and tumors ( f , bottom). Tumors were stained for PBRM1 and p21 expression via IHC analysis ( h ). Scale bar: 200 μm. Source data are provided as a Source Data file.
    Figure Legend Snippet: Mutation of BD4 abolishes the tumor suppressive function of PBRM1. a , b GFP, wild-type or BD4* mutant PBRM1 were stably expressed in Ren-01 PBRM1 KO cells (combination of three clones) ( a ) or PBRM1-null SLR24 cells ( b ). Cells were treated with 50 μM etoposide for the indicated times, and lysates were analyzed via immunoblots with indicated antibodies. The band intensity of indicated protein was measured with Bio-rad Image Lab 4.1, and the relative ratios were calculated over the signal intensity of Vinculin in the corresponding lanes. c – e . Nude mice xenograft analyses were performed with Ren-01 PBRM1 KO cells expressing GFP (left flank) or PBRM1 (right flank). Representative photographs of a mouse ( c , top) and tumors ( c , bottom). Tumors were excised and weighed, and data are presented as mean ± SEM ( d ). p-values were calculated using the paired two-tailed Student’s t -test. Tumors were stained for PBRM1 and p21 expression via IHC ( e ). Scale bar: 200 μm. f – h Nude mice xenograft analyses were performed with Ren-01 PBRM1 KO cells expressing wild-type PBRM1 (left flank) or the BD4* mutant PBRM1 (right flank). Tumors were excised and weighed, and data are presented as mean ± SEM ( g ). p -values were calculated using the paired two-tailed Student’s t -test. Representative photographs of a mouse ( f , top) and tumors ( f , bottom). Tumors were stained for PBRM1 and p21 expression via IHC analysis ( h ). Scale bar: 200 μm. Source data are provided as a Source Data file.

    Techniques Used: Mutagenesis, Stable Transfection, Western Blot, Mouse Assay, Expressing, Two Tailed Test, Staining, Immunohistochemistry

    PBRM1 and p53 display a physical association that is enhanced after DNA damage. a U2OS cells (left) and HEK293T cells (right) were transfected with vector or Flag-PBRM1 and harvested for immunoprecipitation with Flag-M2 beads and elution with 3X Flag peptide. Inputs and eluates were analyzed by immunoblots. b , c H1299 cells were transfected with Flag-PBRM1 and Myc-p53 and treated with etoposide (50 μM, b ) or bleomycin (10 μg/ml, c ) for the indicated times. Lysates were subjected to immunoprecipitation with Flag-M2 beads. Inputs and eluates were analyzed by immunoblots. d HEK293 cells were treated with vehicle (DMSO) or etoposide (50 μM) for 8 h and harvested for immunoprecipitation with control IgG and p53 antibodies. The bound PBRM1 and p53 were examined by immunoblots. e HCT116 and HCT116 p53−/− cells were treated with DMSO or 50 μM etoposide for 24 h. Lysates were immunoprecipitated with p53 antibody. The bound PBRM1 and p53 were examined by immunoblots. Source data are provided as a Source Data file.
    Figure Legend Snippet: PBRM1 and p53 display a physical association that is enhanced after DNA damage. a U2OS cells (left) and HEK293T cells (right) were transfected with vector or Flag-PBRM1 and harvested for immunoprecipitation with Flag-M2 beads and elution with 3X Flag peptide. Inputs and eluates were analyzed by immunoblots. b , c H1299 cells were transfected with Flag-PBRM1 and Myc-p53 and treated with etoposide (50 μM, b ) or bleomycin (10 μg/ml, c ) for the indicated times. Lysates were subjected to immunoprecipitation with Flag-M2 beads. Inputs and eluates were analyzed by immunoblots. d HEK293 cells were treated with vehicle (DMSO) or etoposide (50 μM) for 8 h and harvested for immunoprecipitation with control IgG and p53 antibodies. The bound PBRM1 and p53 were examined by immunoblots. e HCT116 and HCT116 p53−/− cells were treated with DMSO or 50 μM etoposide for 24 h. Lysates were immunoprecipitated with p53 antibody. The bound PBRM1 and p53 were examined by immunoblots. Source data are provided as a Source Data file.

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

    26) Product Images from "Interleukin-1α associates with the tumor suppressor p53 following DNA damage"

    Article Title: Interleukin-1α associates with the tumor suppressor p53 following DNA damage

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-63779-x

    Endogenous IL-1α and p53 coimmunoprecipitate from U2OS cells upon etoposide treatment. Cells were either treated with etoposide (10 µg/ml, 2 hours, +eto) or left untreated (-eto). Coimmunoprecipitation (IP) was performed with anti-p53 antibody (CM-1) and resulting protein samples were split and analysed for IL-1α and p53 content by western blotting in parallel. The IL-1α-specific band is clearly visible in IP from etoposide-treated cells. Asterisk marks an antibody heavy chain. Numbers on the side correspond to the protein MW ladder. Mock IPs were performed from the same amount of the corresponding lysates without adding the CM-1 antibody. For the full-size membrane records we refer to the Supplementary Fig. 7a,b . Similar results we obtained also from A375 cells (Supplementary Fig. 7c ). Supplementary Fig. 8 documents efficient imunoprecipitation of the p53 protein from the lysates.
    Figure Legend Snippet: Endogenous IL-1α and p53 coimmunoprecipitate from U2OS cells upon etoposide treatment. Cells were either treated with etoposide (10 µg/ml, 2 hours, +eto) or left untreated (-eto). Coimmunoprecipitation (IP) was performed with anti-p53 antibody (CM-1) and resulting protein samples were split and analysed for IL-1α and p53 content by western blotting in parallel. The IL-1α-specific band is clearly visible in IP from etoposide-treated cells. Asterisk marks an antibody heavy chain. Numbers on the side correspond to the protein MW ladder. Mock IPs were performed from the same amount of the corresponding lysates without adding the CM-1 antibody. For the full-size membrane records we refer to the Supplementary Fig. 7a,b . Similar results we obtained also from A375 cells (Supplementary Fig. 7c ). Supplementary Fig. 8 documents efficient imunoprecipitation of the p53 protein from the lysates.

    Techniques Used: Western Blot

    Etoposide did not induce colocalization of IL-1α and γ-H2AX in the nucleus of U2OS cells. Cells were treated with etoposide (10 µg/ml, 2 hours) or left untreated. Upper panels show staining for endogenous γ-H2AX (red) and IL-1α (green); lower panels demonstrate staining for endogenous γ-H2AX (red) and p53 (green). Nuclei were stained with DAPI. The intensity profiles show the fluorescence intensity of both labeled proteins in the regions delineated by white lines. Etoposide increases relocalization of IL-1α into cell nuclei and induces formation of both IL-1α and γ-H2AX foci. Arrows above the white line in the γ–H2AX and IL-1α merge image and above the corresponding intensity profile show larger IL-1α foci depleted of the γ-H2AX signal. Images were captured with a Leica SP8 confocal microscope. Scale bars represent 20 µm.
    Figure Legend Snippet: Etoposide did not induce colocalization of IL-1α and γ-H2AX in the nucleus of U2OS cells. Cells were treated with etoposide (10 µg/ml, 2 hours) or left untreated. Upper panels show staining for endogenous γ-H2AX (red) and IL-1α (green); lower panels demonstrate staining for endogenous γ-H2AX (red) and p53 (green). Nuclei were stained with DAPI. The intensity profiles show the fluorescence intensity of both labeled proteins in the regions delineated by white lines. Etoposide increases relocalization of IL-1α into cell nuclei and induces formation of both IL-1α and γ-H2AX foci. Arrows above the white line in the γ–H2AX and IL-1α merge image and above the corresponding intensity profile show larger IL-1α foci depleted of the γ-H2AX signal. Images were captured with a Leica SP8 confocal microscope. Scale bars represent 20 µm.

    Techniques Used: Staining, Fluorescence, Labeling, Microscopy

    Effect of etoposide treatment on the p53•IL-1α interaction, as detected by in situ proximity ligation assay (PLA). PLA was performed with mouse anti-IL-1α and rabbit anti-p53 antibodies in U2OS cells treated with 10 μg/ml etoposide for two hours (n = 364) or control untreated cells (n = 321). ( a ) Representative fluorescence microscopy images used for subsequent analysis of the PLA intensity in nuclei and to compute I n ; etoposide-treated cells show a considerably higher PLA intensity in their nuclei in comparison with untreated cells. ( b ) PLA intensities in the nuclei of cells treated with etoposide were higher than those in untreated cells. The statistical significance of the results was evaluated by Fisher’s exact test (***p
    Figure Legend Snippet: Effect of etoposide treatment on the p53•IL-1α interaction, as detected by in situ proximity ligation assay (PLA). PLA was performed with mouse anti-IL-1α and rabbit anti-p53 antibodies in U2OS cells treated with 10 μg/ml etoposide for two hours (n = 364) or control untreated cells (n = 321). ( a ) Representative fluorescence microscopy images used for subsequent analysis of the PLA intensity in nuclei and to compute I n ; etoposide-treated cells show a considerably higher PLA intensity in their nuclei in comparison with untreated cells. ( b ) PLA intensities in the nuclei of cells treated with etoposide were higher than those in untreated cells. The statistical significance of the results was evaluated by Fisher’s exact test (***p

    Techniques Used: In Situ, Proximity Ligation Assay, Fluorescence, Microscopy

    Etoposide treatment increases intracellular levels of the p53 protein in U2OS and A375 cells. Serial dilutions of cell lysates (load) from untreated cells and cells treated with 10 µg/ml etoposide for two hours were analyzed by western blotting. Membranes were developed with anti-β-actin and anti-p53 (DO-1) antibodies. Numbers on the side correspond to the protein MW ladder.
    Figure Legend Snippet: Etoposide treatment increases intracellular levels of the p53 protein in U2OS and A375 cells. Serial dilutions of cell lysates (load) from untreated cells and cells treated with 10 µg/ml etoposide for two hours were analyzed by western blotting. Membranes were developed with anti-β-actin and anti-p53 (DO-1) antibodies. Numbers on the side correspond to the protein MW ladder.

    Techniques Used: Western Blot

    27) Product Images from "Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA"

    Article Title: Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.02230-18

    Effects of distinct TOP1 and TOP2 inhibitors on HBV cccDNA synthesis. (A) Illustration of DNA TOP2 catalytic cycle. Briefly, TOP2 enzyme binds to the DNA molecule (step 1). In the presence of Mg ++ , two ATP molecules bind to the ATPase domain which results in its dimerization and cleavage of one double-stranded DNA (blue) (step 2). The second DNA molecule (orange) is transported through the break (step 3). Upon transport of the DNA segment through the break, one molecule of the ATP is hydrolyzed (step 4), followed by the religation of the cleaved DNA segment along with hydrolysis of another ATP molecule (step 5) and release of a DNA fragment (step 6). Compounds that inhibit each of these steps are indicated. (B to E) HepAD38 cells were cultured in the absence of Tet, and HBV DNA replication was arrested by PFA treatment between days 3 and 6 after Tet removal. The cells were immediately harvested (0 h) or cultured in the presence of Tet and absence of PFA and mock treated (UT) or treated with 10 μM 3TC or the indicated concentrations of aclarubicin and merbarone (B) or 500 nM ICRF-187, 500 nM etoposide (Etop), 500 nM mitoxantrone (MXT), 500 nM ICRF-193, 500 nM β-lapachone (β-Lap), and 50 μM merbarone (Merb) (E) for 24 h. Hirt DNA was resolved by agarose gel electrophoresis after heat denaturalization at 88°C for 8 min and EcoRI digestion. HBV DNA species were detected by Southern blot hybridization with a riboprobe specifically hybridizing to negative-strand DNA. mtDNA served as a loading control of Hirt DNA analysis. The amounts of cccDNA were quantified by phosphorimager and normalized to the amount of mtDNA. The levels of cccDNA in compound-treated cells were plotted (C and D) or represented as the percentage of that in the mock-treated cells (E).
    Figure Legend Snippet: Effects of distinct TOP1 and TOP2 inhibitors on HBV cccDNA synthesis. (A) Illustration of DNA TOP2 catalytic cycle. Briefly, TOP2 enzyme binds to the DNA molecule (step 1). In the presence of Mg ++ , two ATP molecules bind to the ATPase domain which results in its dimerization and cleavage of one double-stranded DNA (blue) (step 2). The second DNA molecule (orange) is transported through the break (step 3). Upon transport of the DNA segment through the break, one molecule of the ATP is hydrolyzed (step 4), followed by the religation of the cleaved DNA segment along with hydrolysis of another ATP molecule (step 5) and release of a DNA fragment (step 6). Compounds that inhibit each of these steps are indicated. (B to E) HepAD38 cells were cultured in the absence of Tet, and HBV DNA replication was arrested by PFA treatment between days 3 and 6 after Tet removal. The cells were immediately harvested (0 h) or cultured in the presence of Tet and absence of PFA and mock treated (UT) or treated with 10 μM 3TC or the indicated concentrations of aclarubicin and merbarone (B) or 500 nM ICRF-187, 500 nM etoposide (Etop), 500 nM mitoxantrone (MXT), 500 nM ICRF-193, 500 nM β-lapachone (β-Lap), and 50 μM merbarone (Merb) (E) for 24 h. Hirt DNA was resolved by agarose gel electrophoresis after heat denaturalization at 88°C for 8 min and EcoRI digestion. HBV DNA species were detected by Southern blot hybridization with a riboprobe specifically hybridizing to negative-strand DNA. mtDNA served as a loading control of Hirt DNA analysis. The amounts of cccDNA were quantified by phosphorimager and normalized to the amount of mtDNA. The levels of cccDNA in compound-treated cells were plotted (C and D) or represented as the percentage of that in the mock-treated cells (E).

    Techniques Used: Cell Culture, Agarose Gel Electrophoresis, Southern Blot, Hybridization

    28) Product Images from "Identification of Novel Small-Molecule Kinase Modulators for the Treatment of Neuroblastoma"

    Article Title: Identification of Novel Small-Molecule Kinase Modulators for the Treatment of Neuroblastoma

    Journal: Oncology and Therapy

    doi: 10.1007/s40487-020-00113-5

    High-throughput drug screening in the IGR-N-91 neuroblastoma cell line. a Schematic of the screening procedure with the Acti-TargK960 small-molecule library. b Overview of the plates in the final screen, after 72 h of treatment with the compounds at 1 and 10 µM. c Volcano plot of all wells (control, doxorubicin, etoposide and test compounds). Horizontal line corresponds to a false discovery rate ( FDR ) = 0.01, and compounds under this line are considered to be ‘not significant’ in terms of cytotoxic activity. The two vertical lines correspond to a change of 0.5 in cytotoxic activity, and compounds outside this range are considered to have ‘strong’ cytotoxic activity. d Heatmap of the activity of 55 compounds that were considered to have significant and strong cytotoxic activity. Doxorubicin, a chemotherapeutic used to treat neuroblastoma, and dimethyl sulfoxide ( DMSO ) were used as controls
    Figure Legend Snippet: High-throughput drug screening in the IGR-N-91 neuroblastoma cell line. a Schematic of the screening procedure with the Acti-TargK960 small-molecule library. b Overview of the plates in the final screen, after 72 h of treatment with the compounds at 1 and 10 µM. c Volcano plot of all wells (control, doxorubicin, etoposide and test compounds). Horizontal line corresponds to a false discovery rate ( FDR ) = 0.01, and compounds under this line are considered to be ‘not significant’ in terms of cytotoxic activity. The two vertical lines correspond to a change of 0.5 in cytotoxic activity, and compounds outside this range are considered to have ‘strong’ cytotoxic activity. d Heatmap of the activity of 55 compounds that were considered to have significant and strong cytotoxic activity. Doxorubicin, a chemotherapeutic used to treat neuroblastoma, and dimethyl sulfoxide ( DMSO ) were used as controls

    Techniques Used: High Throughput Screening Assay, Activity Assay

    29) Product Images from "?H2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments"

    Article Title: ?H2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-483

    Cellular biomarker responses in HT29 cells exposed to various cytotoxic chemotherapeutic agents in combination with the Chk1 inhibitor V158411. HT29 cells were exposed to the combination GI 80 of gemcitabine (0.2 μM), camptothecin (0.44 μM), cisplatin (68 μM), oxaliplatin (131 μM), doxorubicin (1.2 μM) or etoposide (59 μM) for 18 hours followed by DMSO (−) or 400 nM V158411 (+) for a further 24 hours. Protein expression was characterized by immunoblotting.
    Figure Legend Snippet: Cellular biomarker responses in HT29 cells exposed to various cytotoxic chemotherapeutic agents in combination with the Chk1 inhibitor V158411. HT29 cells were exposed to the combination GI 80 of gemcitabine (0.2 μM), camptothecin (0.44 μM), cisplatin (68 μM), oxaliplatin (131 μM), doxorubicin (1.2 μM) or etoposide (59 μM) for 18 hours followed by DMSO (−) or 400 nM V158411 (+) for a further 24 hours. Protein expression was characterized by immunoblotting.

    Techniques Used: Biomarker Assay, Expressing

    Checkpoint activation and DNA damage protein biomarker responses in HT29 cells following treatment with cytotoxic chemotherapeutic agents. HT29 cells were treated with approximately 5-times the single agent GI 50 of gemcitabine (Gem, 0.2 μM), camptothecin (CPT, 1 μM), cisplatin (CP, 125 μM), oxaliplatin (OxPt, 250 μM), doxorubicin (Dox, 3 μM) or etoposide (Etop, 50 μM) for 24 hours. Changes in protein expression levels were determined by immunoblotting.
    Figure Legend Snippet: Checkpoint activation and DNA damage protein biomarker responses in HT29 cells following treatment with cytotoxic chemotherapeutic agents. HT29 cells were treated with approximately 5-times the single agent GI 50 of gemcitabine (Gem, 0.2 μM), camptothecin (CPT, 1 μM), cisplatin (CP, 125 μM), oxaliplatin (OxPt, 250 μM), doxorubicin (Dox, 3 μM) or etoposide (Etop, 50 μM) for 24 hours. Changes in protein expression levels were determined by immunoblotting.

    Techniques Used: Activation Assay, Biomarker Assay, Cycling Probe Technology, Expressing

    V158411 inhibits DNA damage induced Chk1 auto-phosphorylation and increases γH2AX in colon carcinoma cells. HT29 (left) or Colo205 (right) p53 defective colon carcinoma cells were treated with A . 200 nM camptothecin or 100 nM gemcitabine plus varying concentrations of V158411 for 24 hours or B . treated with the indicated concentrations of gemcitabine, camptothecin, cisplatin or etoposide with either DMSO or 200 nM V158411 for 24 hours. Protein expression was characterized by immunoblotting.
    Figure Legend Snippet: V158411 inhibits DNA damage induced Chk1 auto-phosphorylation and increases γH2AX in colon carcinoma cells. HT29 (left) or Colo205 (right) p53 defective colon carcinoma cells were treated with A . 200 nM camptothecin or 100 nM gemcitabine plus varying concentrations of V158411 for 24 hours or B . treated with the indicated concentrations of gemcitabine, camptothecin, cisplatin or etoposide with either DMSO or 200 nM V158411 for 24 hours. Protein expression was characterized by immunoblotting.

    Techniques Used: Expressing

    30) Product Images from "Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells"

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0493-5

    ALDH Bright cells are resistant to BTZ but sensitive to DSF/Cu 2+ , and BTZ-resistance confers increased resistance to Ara-C in CMY cells. CMY cells were treated with 5 nM BTZ and processed for ALDH activity using ALDELUOR TM assay and flow cytometry. Dot plot shows the percentage of ALDH-positive cells (FITC) on the x-axis, and the sideward scatter (SSC-A) on the y-axis. The gated cell populations were created using the ALDH inhibitor DEAB provided with the kit. One percent of the ALDH Bright cell population was resistant to BTZ ( a ). Because these cells may play a role in BTZ resistance, this subpopulation was flow-sorted and subsequently subjected to drug dose response assays of either DSF/Cu 2+ or a re-exposure to BTZ. The BTZ-resistant ALDH bright subpopulation of CMY cells was still resistant to BTZ (IC 50 7.7 nM) compared to untreated, unsorted CMY cells (IC 50 2 nM), but were sensitive to DSF/Cu 2+ (IC 50 56 nM vs 52 nM for untreated, unsorted CMY cells) ( b ). BTZ-resistant variants from CMY and CMK cell lines (CMY-BR and CMK-BR) were generated by exposure to stepwise increasing concentrations of BTZ up to 200 nM for CMY, and 100 nM for CMK. These cells were treated with different doses of Ara-C, VP-16 and daunorubicin. Dose response curves were plotted in comparison to the CMY and CMK parent cell lines. Approximately 25% of the CMY-BR cells remained viable after treatment with high Ara-C doses for 72 h, but the parental cells completely died at the same doses. The CMK cell line and its BTZ-resistant variant (CMK-BR) were both equally sensitive to Ara-C, VP-16 and daunorubicin ( c )
    Figure Legend Snippet: ALDH Bright cells are resistant to BTZ but sensitive to DSF/Cu 2+ , and BTZ-resistance confers increased resistance to Ara-C in CMY cells. CMY cells were treated with 5 nM BTZ and processed for ALDH activity using ALDELUOR TM assay and flow cytometry. Dot plot shows the percentage of ALDH-positive cells (FITC) on the x-axis, and the sideward scatter (SSC-A) on the y-axis. The gated cell populations were created using the ALDH inhibitor DEAB provided with the kit. One percent of the ALDH Bright cell population was resistant to BTZ ( a ). Because these cells may play a role in BTZ resistance, this subpopulation was flow-sorted and subsequently subjected to drug dose response assays of either DSF/Cu 2+ or a re-exposure to BTZ. The BTZ-resistant ALDH bright subpopulation of CMY cells was still resistant to BTZ (IC 50 7.7 nM) compared to untreated, unsorted CMY cells (IC 50 2 nM), but were sensitive to DSF/Cu 2+ (IC 50 56 nM vs 52 nM for untreated, unsorted CMY cells) ( b ). BTZ-resistant variants from CMY and CMK cell lines (CMY-BR and CMK-BR) were generated by exposure to stepwise increasing concentrations of BTZ up to 200 nM for CMY, and 100 nM for CMK. These cells were treated with different doses of Ara-C, VP-16 and daunorubicin. Dose response curves were plotted in comparison to the CMY and CMK parent cell lines. Approximately 25% of the CMY-BR cells remained viable after treatment with high Ara-C doses for 72 h, but the parental cells completely died at the same doses. The CMK cell line and its BTZ-resistant variant (CMK-BR) were both equally sensitive to Ara-C, VP-16 and daunorubicin ( c )

    Techniques Used: Acetylene Reduction Assay, Activity Assay, Flow Cytometry, Cytometry, Generated, Variant Assay

    31) Product Images from "Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer"

    Article Title: Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-017-1197-5

    c.1380 CDH1 SB.mhdgc-1 gastric cancer cells show vulnerabilities to toposisomerase II and PI3K/mTOR inhibition. a Drug response curves of a panel of gastric cancer cell lines treated with a range of concentrations of mitoxantrone, etoposide (both TOPO2A inhibitors), or PI-103 (dual class IA phosphatidylinositol 3 kinase/mTOR inhibitor) for 72 h. X-axis indicates log [concentration] tested, y-axis indicates cell viability percentage normalized to vehicle-control samples. Mean cell viability values are plotted with standard error of the mean (SEM) from at least 2 independent experiments done in triplicate. b Rate of apoptosis induced by 24-h treatment of 1 µM etoposide, mitoxantrone, or PI-103 normalized to DMSO-treated samples in sporadic gastric cancer SNU-16 and hereditary c.1380delA SB.mhdgc-1 cells. Flow cytometry profiles of FITC-labeled anti-BrdU staining of 3′-hydroxyl (OH) termini of double- and single-stranded DNA, relative BrdU fractions normalized to DMSO-treated control shown on the right
    Figure Legend Snippet: c.1380 CDH1 SB.mhdgc-1 gastric cancer cells show vulnerabilities to toposisomerase II and PI3K/mTOR inhibition. a Drug response curves of a panel of gastric cancer cell lines treated with a range of concentrations of mitoxantrone, etoposide (both TOPO2A inhibitors), or PI-103 (dual class IA phosphatidylinositol 3 kinase/mTOR inhibitor) for 72 h. X-axis indicates log [concentration] tested, y-axis indicates cell viability percentage normalized to vehicle-control samples. Mean cell viability values are plotted with standard error of the mean (SEM) from at least 2 independent experiments done in triplicate. b Rate of apoptosis induced by 24-h treatment of 1 µM etoposide, mitoxantrone, or PI-103 normalized to DMSO-treated samples in sporadic gastric cancer SNU-16 and hereditary c.1380delA SB.mhdgc-1 cells. Flow cytometry profiles of FITC-labeled anti-BrdU staining of 3′-hydroxyl (OH) termini of double- and single-stranded DNA, relative BrdU fractions normalized to DMSO-treated control shown on the right

    Techniques Used: Inhibition, IA, Concentration Assay, Flow Cytometry, Cytometry, Labeling, BrdU Staining

    32) Product Images from "Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis"

    Article Title: Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02950-5

    Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments
    Figure Legend Snippet: Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m 2 ) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g , h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments

    Techniques Used: Irradiation, Immunoprecipitation, Sequencing, Concentration Assay, Purification, Knock-Out, Transfection, Activity Assay, FLAG-tag, Incubation, Recombinant, Acetylation Assay, Staining

    33) Product Images from "Novel ADAM-17 inhibitor ZLDI-8 enhances the in vitro and in vivo chemotherapeutic effects of Sorafenib on hepatocellular carcinoma cells"

    Article Title: Novel ADAM-17 inhibitor ZLDI-8 enhances the in vitro and in vivo chemotherapeutic effects of Sorafenib on hepatocellular carcinoma cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0804-6

    Effect of ZLDI-8 on cytotoxic activity of Etoposide or paclitaxel on MHCC97-H cell-cycle arrest. Cells, were pre-treated with ZLDI-8, were treated with IC 50 concentration of Etoposide ( a ) or Paclitaxel ( b ). Then, the cells were harvested and flow cytometer was performed. Results were shown as photographs or mean ± SD. * p
    Figure Legend Snippet: Effect of ZLDI-8 on cytotoxic activity of Etoposide or paclitaxel on MHCC97-H cell-cycle arrest. Cells, were pre-treated with ZLDI-8, were treated with IC 50 concentration of Etoposide ( a ) or Paclitaxel ( b ). Then, the cells were harvested and flow cytometer was performed. Results were shown as photographs or mean ± SD. * p

    Techniques Used: Activity Assay, Concentration Assay, Flow Cytometry, Cytometry

    34) Product Images from "PTEN Methylation by NSD2 Controls Cellular Sensitivity to DNA Damage"

    Article Title: PTEN Methylation by NSD2 Controls Cellular Sensitivity to DNA Damage

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-18-0083

    ATM-mediated phosphorylation of PTEN is required for its binding with the BRCT domain of MDC1 upon DNA damage signaling. (A) (pS/TQ) upon etoposide treatment. Immunoblot (IB) analysis of anti-Pten immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treated with 30 μM etoposide as indicated time points before harvesting. (B) Etoposide treatment promoted PTEN interaction with MDC1 BRCT domain, but neither MDC1 FHA nor 53BP1 BRCT domain. IB analysis of GST pull-down and WCL derived from U2OS cells transfected with indicated constructs and treatment with/without 30 μM etoposide for 30 min before harvesting. (C) Etoposide treatment promoted wild type (WT), but not T398A mutant PTEN, interaction with MDC1 BRCT domain. IB analysis of GST pull-down and WCL derived from U2OS cells co-transfected with indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min and harvested for IP assays. (D) PTEN, MDC1, and NSD2 formed a tertiary complex in the nucleus upon etoposide treatment. IB analysis of anti-PTEN IPs from cytoplasm or nucleus as well as WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (E) Depletion of ATM disrupted PTEN interaction with MDC1 and NSD2 upon etoposide treatment. IB analysis of anti-PTEN IPs and WCL derived from U2OS cell lines stably expressing shControl or shATM transfected with indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min before harvesting. (F) Depletion of Mdc1 impaired the interaction between Pten and Nsd2. IB analysis of anti-Pten IPs and WCL derived from Mdc1 +/+ and Mdc1 −/− MEFs treated with 30 μM etoposide as indicated time points before harvesting.
    Figure Legend Snippet: ATM-mediated phosphorylation of PTEN is required for its binding with the BRCT domain of MDC1 upon DNA damage signaling. (A) (pS/TQ) upon etoposide treatment. Immunoblot (IB) analysis of anti-Pten immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treated with 30 μM etoposide as indicated time points before harvesting. (B) Etoposide treatment promoted PTEN interaction with MDC1 BRCT domain, but neither MDC1 FHA nor 53BP1 BRCT domain. IB analysis of GST pull-down and WCL derived from U2OS cells transfected with indicated constructs and treatment with/without 30 μM etoposide for 30 min before harvesting. (C) Etoposide treatment promoted wild type (WT), but not T398A mutant PTEN, interaction with MDC1 BRCT domain. IB analysis of GST pull-down and WCL derived from U2OS cells co-transfected with indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min and harvested for IP assays. (D) PTEN, MDC1, and NSD2 formed a tertiary complex in the nucleus upon etoposide treatment. IB analysis of anti-PTEN IPs from cytoplasm or nucleus as well as WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (E) Depletion of ATM disrupted PTEN interaction with MDC1 and NSD2 upon etoposide treatment. IB analysis of anti-PTEN IPs and WCL derived from U2OS cell lines stably expressing shControl or shATM transfected with indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min before harvesting. (F) Depletion of Mdc1 impaired the interaction between Pten and Nsd2. IB analysis of anti-Pten IPs and WCL derived from Mdc1 +/+ and Mdc1 −/− MEFs treated with 30 μM etoposide as indicated time points before harvesting.

    Techniques Used: Binding Assay, Derivative Assay, Transfection, Construct, Mutagenesis, Stable Transfection, Expressing

    NSD2-mediated di-methylation of PTEN is recognized by the tudor domain of 53BP1. (A) Etoposide treatment enhanced PTEN interaction with 53BP1. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (B and C) PTEN-K349R mutant disrupted its binding with 53BP1 upon etoposide treatment. IB analysis of GST pull-down and WCL derived from U2OS cells transfected with the indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min and harvested for GST pull-down assays. (D) 53BP1 tudor domain had a high affinity with K349-me2-PTEN peptides. 1 μg of indicated biotin-labeled synthetic PTEN peptides were incubated with 250 ng purified recombinant GST-tagged 53BP1 tudor domain, respectively. Streptavidin beads were added to perform pull-down assays and precipitations were analyzed by IB. Dot blot assays were performed to show equal amount of biotinylated peptides was used for the pull-down assay. (E and F) Depletion of NSD2 disrupted PTEN interaction with 53BP1. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells stably expressing shNSD2 ( E ) or Nsd2 −/− MEFs ( F ) that were treated with/without 30 μM etoposide for 30 min before harvesting.
    Figure Legend Snippet: NSD2-mediated di-methylation of PTEN is recognized by the tudor domain of 53BP1. (A) Etoposide treatment enhanced PTEN interaction with 53BP1. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (B and C) PTEN-K349R mutant disrupted its binding with 53BP1 upon etoposide treatment. IB analysis of GST pull-down and WCL derived from U2OS cells transfected with the indicated constructs. 36 h after transfection, cells were treated with/without 30 μM etoposide for 30 min and harvested for GST pull-down assays. (D) 53BP1 tudor domain had a high affinity with K349-me2-PTEN peptides. 1 μg of indicated biotin-labeled synthetic PTEN peptides were incubated with 250 ng purified recombinant GST-tagged 53BP1 tudor domain, respectively. Streptavidin beads were added to perform pull-down assays and precipitations were analyzed by IB. Dot blot assays were performed to show equal amount of biotinylated peptides was used for the pull-down assay. (E and F) Depletion of NSD2 disrupted PTEN interaction with 53BP1. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells stably expressing shNSD2 ( E ) or Nsd2 −/− MEFs ( F ) that were treated with/without 30 μM etoposide for 30 min before harvesting.

    Techniques Used: Methylation, Derivative Assay, Mutagenesis, Binding Assay, Transfection, Construct, Labeling, Incubation, Purification, Recombinant, Dot Blot, Pull Down Assay, Stable Transfection, Expressing

    DNA damage promotes NSD2-mediated di-methylation of PTEN at K349. (A) . IB analysis of anti-PTEN IPs and WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (B) Depletion of Nsd2 impaired the di-methylation of Pten. IB analysis of anti-Pten IPs and WCL derived from Nsd2 +/+ and Nsd2 −/− MEFs treated with 30 μM etoposide for 30 min before harvesting. (C) K349 was identified as the major di-methylation site on PTEN. IB analysis of anti-HA IPs and WCL derived from 293T cells transfected with the indicated constructs and treated with/without 30 μM etoposide for 30 min before harvesting. (D) PTEN was detected by the specific K349 di-methylation (K349me2) antibody. IB analysis of anti-HA IPs and WCL derived from U2OS cells transfected with HA-PTEN WT or K349R mutant and treated with/without 30 μM etoposide at indicated time points. (E) The K349 di-methylation of Pten existed in both cytoplasm and nucleus. IB analysis of anti-Pten IPs from cytoplasm or nucleus as well as WCL derived from NIH3T3 cells treatment with 30 μM etoposide as indicated time points before harvesting. (F and G) NSD2 deficiency decreased the di-methylation of PTEN at K349. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells stably expressing shNSD2 ( F ) or Nsd2 −/− MEFs ( G ) that were treated with IR (5 Gy) at indicated time points before harvesting.
    Figure Legend Snippet: DNA damage promotes NSD2-mediated di-methylation of PTEN at K349. (A) . IB analysis of anti-PTEN IPs and WCL derived from U2OS cells treated with 30 μM etoposide as indicated time points before harvesting. (B) Depletion of Nsd2 impaired the di-methylation of Pten. IB analysis of anti-Pten IPs and WCL derived from Nsd2 +/+ and Nsd2 −/− MEFs treated with 30 μM etoposide for 30 min before harvesting. (C) K349 was identified as the major di-methylation site on PTEN. IB analysis of anti-HA IPs and WCL derived from 293T cells transfected with the indicated constructs and treated with/without 30 μM etoposide for 30 min before harvesting. (D) PTEN was detected by the specific K349 di-methylation (K349me2) antibody. IB analysis of anti-HA IPs and WCL derived from U2OS cells transfected with HA-PTEN WT or K349R mutant and treated with/without 30 μM etoposide at indicated time points. (E) The K349 di-methylation of Pten existed in both cytoplasm and nucleus. IB analysis of anti-Pten IPs from cytoplasm or nucleus as well as WCL derived from NIH3T3 cells treatment with 30 μM etoposide as indicated time points before harvesting. (F and G) NSD2 deficiency decreased the di-methylation of PTEN at K349. IB analysis of anti-PTEN IPs and WCL derived from U2OS cells stably expressing shNSD2 ( F ) or Nsd2 −/− MEFs ( G ) that were treated with IR (5 Gy) at indicated time points before harvesting.

    Techniques Used: Methylation, Derivative Assay, Transfection, Construct, Mutagenesis, Stable Transfection, Expressing

    35) Product Images from "Targeting Poly (ADP-Ribose) Polymerase Partially Contributes to Bufalin-Induced Cell Death in Multiple Myeloma Cells"

    Article Title: Targeting Poly (ADP-Ribose) Polymerase Partially Contributes to Bufalin-Induced Cell Death in Multiple Myeloma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066130

    Bufalin enhances chemosensitivity of H929 cells. (A–D) H929 cells were treated with 10 nM bufalin alone, or in combination with 50 nM topotecan, 10 nM camptothecin, 2.5 µM etoposide, and 0.5 µM vorinostat, respectively for 48 h. Cell proliferation was measured by CCK8 assay. All values represent means ± S.D. of three independent experiments, each performed in triplicate (. ** P
    Figure Legend Snippet: Bufalin enhances chemosensitivity of H929 cells. (A–D) H929 cells were treated with 10 nM bufalin alone, or in combination with 50 nM topotecan, 10 nM camptothecin, 2.5 µM etoposide, and 0.5 µM vorinostat, respectively for 48 h. Cell proliferation was measured by CCK8 assay. All values represent means ± S.D. of three independent experiments, each performed in triplicate (. ** P

    Techniques Used: CCK-8 Assay

    36) Product Images from "Multiparametric Cell Cycle Analysis Using the Operetta High-Content Imager and Harmony Software with PhenoLOGIC"

    Article Title: Multiparametric Cell Cycle Analysis Using the Operetta High-Content Imager and Harmony Software with PhenoLOGIC

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134306

    Validation of image analysis protocol. HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU and pHH3 (S10). (A) Example histograms following treatment with DMSO, etoposide, paclitaxel or VX680. Cell cycle distribution was determined using either DNA histogram analysis (B) or multiparametric analysis (C). (D) Time course and dose response of cell cycle changes determined using the multiparametric method for cells treated with camptothecin, gemcitabine or etoposide. (E) Proliferation index (EdU positive cells treated / EdU positive cells control) and cell number were determined from the images. Values are the average of 6 technical replicates.
    Figure Legend Snippet: Validation of image analysis protocol. HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU and pHH3 (S10). (A) Example histograms following treatment with DMSO, etoposide, paclitaxel or VX680. Cell cycle distribution was determined using either DNA histogram analysis (B) or multiparametric analysis (C). (D) Time course and dose response of cell cycle changes determined using the multiparametric method for cells treated with camptothecin, gemcitabine or etoposide. (E) Proliferation index (EdU positive cells treated / EdU positive cells control) and cell number were determined from the images. Values are the average of 6 technical replicates.

    Techniques Used: Staining

    Determination of cell cycle phase of γH2AX positive cells following treatment with a cytotoxic DNA damaging agent. HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU, pHH3 (S10) and pH2AX (S139). (A) Example images from HT29 cells treated with DMSO, camptothecin or etoposide demonstrating the presence of pH2AX (S139) positive (γH2AX) nuclei. (B) Quantification of the fraction of γH2AX positive nuclei following treatment with the various agents. (C) Cell cycle distribution of γH2AX positive nuclei following multiparametric analysis. Values are the average of 6 technical replicates ± SD.
    Figure Legend Snippet: Determination of cell cycle phase of γH2AX positive cells following treatment with a cytotoxic DNA damaging agent. HT29 cells were treated with the indicated compound for 24 hour and then fixed and stained with Hoechst 33342, EdU, pHH3 (S10) and pH2AX (S139). (A) Example images from HT29 cells treated with DMSO, camptothecin or etoposide demonstrating the presence of pH2AX (S139) positive (γH2AX) nuclei. (B) Quantification of the fraction of γH2AX positive nuclei following treatment with the various agents. (C) Cell cycle distribution of γH2AX positive nuclei following multiparametric analysis. Values are the average of 6 technical replicates ± SD.

    Techniques Used: Staining

    37) Product Images from "Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer"

    Article Title: Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-017-1197-5

    c.1380 CDH1 SB.mhdgc-1 gastric cancer cells show vulnerabilities to toposisomerase II and PI3K/mTOR inhibition. a Drug response curves of a panel of gastric cancer cell lines treated with a range of concentrations of mitoxantrone, etoposide (both TOPO2A inhibitors), or PI-103 (dual class IA phosphatidylinositol 3 kinase/mTOR inhibitor) for 72 h. X-axis indicates log [concentration] tested, y-axis indicates cell viability percentage normalized to vehicle-control samples. Mean cell viability values are plotted with standard error of the mean (SEM) from at least 2 independent experiments done in triplicate. b Rate of apoptosis induced by 24-h treatment of 1 µM etoposide, mitoxantrone, or PI-103 normalized to DMSO-treated samples in sporadic gastric cancer SNU-16 and hereditary c.1380delA SB.mhdgc-1 cells. Flow cytometry profiles of FITC-labeled anti-BrdU staining of 3′-hydroxyl (OH) termini of double- and single-stranded DNA, relative BrdU fractions normalized to DMSO-treated control shown on the right
    Figure Legend Snippet: c.1380 CDH1 SB.mhdgc-1 gastric cancer cells show vulnerabilities to toposisomerase II and PI3K/mTOR inhibition. a Drug response curves of a panel of gastric cancer cell lines treated with a range of concentrations of mitoxantrone, etoposide (both TOPO2A inhibitors), or PI-103 (dual class IA phosphatidylinositol 3 kinase/mTOR inhibitor) for 72 h. X-axis indicates log [concentration] tested, y-axis indicates cell viability percentage normalized to vehicle-control samples. Mean cell viability values are plotted with standard error of the mean (SEM) from at least 2 independent experiments done in triplicate. b Rate of apoptosis induced by 24-h treatment of 1 µM etoposide, mitoxantrone, or PI-103 normalized to DMSO-treated samples in sporadic gastric cancer SNU-16 and hereditary c.1380delA SB.mhdgc-1 cells. Flow cytometry profiles of FITC-labeled anti-BrdU staining of 3′-hydroxyl (OH) termini of double- and single-stranded DNA, relative BrdU fractions normalized to DMSO-treated control shown on the right

    Techniques Used: Inhibition, IA, Concentration Assay, Flow Cytometry, Cytometry, Labeling, BrdU Staining

    38) Product Images from "Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma"

    Article Title: Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171066

    ATSP-7041 synergizes with chemotherapy agents . (A–C­) CI plots for the combination of ATSP-7041 with doxorubicin, etoposide, and vincristine after 3-d treatment of (A) TC32, (B) TC138, and (C) CHLA258 cells. (D) Western blots showing increased p53 protein levels in TC32 cells treat with combinations of ATSP-7041 and doxorubicin. Cells were treated at indicated concentrations for 2 d (ATSP, ATSP-7041; Doxo, doxorubicin). (E) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and etoposide. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Eto, etoposide). (F) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and vincristine. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Vinc, vincristine).
    Figure Legend Snippet: ATSP-7041 synergizes with chemotherapy agents . (A–C­) CI plots for the combination of ATSP-7041 with doxorubicin, etoposide, and vincristine after 3-d treatment of (A) TC32, (B) TC138, and (C) CHLA258 cells. (D) Western blots showing increased p53 protein levels in TC32 cells treat with combinations of ATSP-7041 and doxorubicin. Cells were treated at indicated concentrations for 2 d (ATSP, ATSP-7041; Doxo, doxorubicin). (E) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and etoposide. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Eto, etoposide). (F) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and vincristine. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Vinc, vincristine).

    Techniques Used: Western Blot

    Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (A) Western blots showing attenuated increase of p53 protein levels in TC32, TC138, and CHLA258 cells infected with sgRNAs targeting TP53 after etoposide treatment (Control, control sgRNA; sg #1, sg TP53 #1; sg #2, sg TP53 #2; sg #4, sg TP53 #4; sg #5, sg TP53 #5). Cells were treated with vehicle or 50 μM etoposide for one hour (Veh, vehicle; Eto, etoposide). (B) TP53 knockout cells were treated with ATSP-7041 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (C) TP53 knockout cells were treated with GSK2830371 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (D) TP53 knockout cells were treated with P5091 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (E) Western blots showing decreased protein levels of USP7 after infection with sgRNAs targeting USP7 in TC32 TP53 knockout cells. (F) Western blots showing decreased protein levels of Wip1 after infection with sgRNAs targeting PPM1D in TC32 TP53 knockout cells. (G) Relative viability of TC32 TP53 knockout cells infected with sgRNAs targeting USP7 or PPM1D or control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. Significance was calculated by paired, two-tailed t test: not significant (n.s.) for P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) Ewing sarcoma cells were treated with XL-188 for 3 d. TP53 wild-type Ewing sarcoma cell lines are shown in red. TP53 mutated Ewing sarcoma cell lines are shown in black. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (I) TP53 knockout cells were treated with XL-188 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown.
    Figure Legend Snippet: Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (A) Western blots showing attenuated increase of p53 protein levels in TC32, TC138, and CHLA258 cells infected with sgRNAs targeting TP53 after etoposide treatment (Control, control sgRNA; sg #1, sg TP53 #1; sg #2, sg TP53 #2; sg #4, sg TP53 #4; sg #5, sg TP53 #5). Cells were treated with vehicle or 50 μM etoposide for one hour (Veh, vehicle; Eto, etoposide). (B) TP53 knockout cells were treated with ATSP-7041 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (C) TP53 knockout cells were treated with GSK2830371 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (D) TP53 knockout cells were treated with P5091 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (E) Western blots showing decreased protein levels of USP7 after infection with sgRNAs targeting USP7 in TC32 TP53 knockout cells. (F) Western blots showing decreased protein levels of Wip1 after infection with sgRNAs targeting PPM1D in TC32 TP53 knockout cells. (G) Relative viability of TC32 TP53 knockout cells infected with sgRNAs targeting USP7 or PPM1D or control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. Significance was calculated by paired, two-tailed t test: not significant (n.s.) for P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) Ewing sarcoma cells were treated with XL-188 for 3 d. TP53 wild-type Ewing sarcoma cell lines are shown in red. TP53 mutated Ewing sarcoma cell lines are shown in black. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (I) TP53 knockout cells were treated with XL-188 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown.

    Techniques Used: Western Blot, Infection, Knock-Out, Standard Deviation, Two Tailed Test

    39) Product Images from "TGF?/TNF?-Mediated Epithelial-Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype"

    Article Title: TGF?/TNF?-Mediated Epithelial-Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-4554

    ETTM stem cells are resistant to oxaliplatin, etoposide and paclitaxel
    Figure Legend Snippet: ETTM stem cells are resistant to oxaliplatin, etoposide and paclitaxel

    Techniques Used:

    40) Product Images from "Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma"

    Article Title: Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171066

    ATSP-7041 synergizes with chemotherapy agents . (A–C­) CI plots for the combination of ATSP-7041 with doxorubicin, etoposide, and vincristine after 3-d treatment of (A) TC32, (B) TC138, and (C) CHLA258 cells. (D) Western blots showing increased p53 protein levels in TC32 cells treat with combinations of ATSP-7041 and doxorubicin. Cells were treated at indicated concentrations for 2 d (ATSP, ATSP-7041; Doxo, doxorubicin). (E) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and etoposide. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Eto, etoposide). (F) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and vincristine. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Vinc, vincristine).
    Figure Legend Snippet: ATSP-7041 synergizes with chemotherapy agents . (A–C­) CI plots for the combination of ATSP-7041 with doxorubicin, etoposide, and vincristine after 3-d treatment of (A) TC32, (B) TC138, and (C) CHLA258 cells. (D) Western blots showing increased p53 protein levels in TC32 cells treat with combinations of ATSP-7041 and doxorubicin. Cells were treated at indicated concentrations for 2 d (ATSP, ATSP-7041; Doxo, doxorubicin). (E) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and etoposide. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Eto, etoposide). (F) Western blots showing increased p53 protein levels in TC32 cells treated with combinations of ATSP-7041 and vincristine. Cells were treated at the indicated concentrations for 2 d (ATSP, ATSP-7041; Vinc, vincristine).

    Techniques Used: Western Blot

    Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (A) Western blots showing attenuated increase of p53 protein levels in TC32, TC138, and CHLA258 cells infected with sgRNAs targeting TP53 after etoposide treatment (Control, control sgRNA; sg #1, sg TP53 #1; sg #2, sg TP53 #2; sg #4, sg TP53 #4; sg #5, sg TP53 #5). Cells were treated with vehicle or 50 μM etoposide for one hour (Veh, vehicle; Eto, etoposide). (B) TP53 knockout cells were treated with ATSP-7041 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (C) TP53 knockout cells were treated with GSK2830371 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (D) TP53 knockout cells were treated with P5091 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (E) Western blots showing decreased protein levels of USP7 after infection with sgRNAs targeting USP7 in TC32 TP53 knockout cells. (F) Western blots showing decreased protein levels of Wip1 after infection with sgRNAs targeting PPM1D in TC32 TP53 knockout cells. (G) Relative viability of TC32 TP53 knockout cells infected with sgRNAs targeting USP7 or PPM1D or control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. Significance was calculated by paired, two-tailed t test: not significant (n.s.) for P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) Ewing sarcoma cells were treated with XL-188 for 3 d. TP53 wild-type Ewing sarcoma cell lines are shown in red. TP53 mutated Ewing sarcoma cell lines are shown in black. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (I) TP53 knockout cells were treated with XL-188 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown.
    Figure Legend Snippet: Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (A) Western blots showing attenuated increase of p53 protein levels in TC32, TC138, and CHLA258 cells infected with sgRNAs targeting TP53 after etoposide treatment (Control, control sgRNA; sg #1, sg TP53 #1; sg #2, sg TP53 #2; sg #4, sg TP53 #4; sg #5, sg TP53 #5). Cells were treated with vehicle or 50 μM etoposide for one hour (Veh, vehicle; Eto, etoposide). (B) TP53 knockout cells were treated with ATSP-7041 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (C) TP53 knockout cells were treated with GSK2830371 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (D) TP53 knockout cells were treated with P5091 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (E) Western blots showing decreased protein levels of USP7 after infection with sgRNAs targeting USP7 in TC32 TP53 knockout cells. (F) Western blots showing decreased protein levels of Wip1 after infection with sgRNAs targeting PPM1D in TC32 TP53 knockout cells. (G) Relative viability of TC32 TP53 knockout cells infected with sgRNAs targeting USP7 or PPM1D or control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. Significance was calculated by paired, two-tailed t test: not significant (n.s.) for P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) Ewing sarcoma cells were treated with XL-188 for 3 d. TP53 wild-type Ewing sarcoma cell lines are shown in red. TP53 mutated Ewing sarcoma cell lines are shown in black. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (I) TP53 knockout cells were treated with XL-188 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown.

    Techniques Used: Western Blot, Infection, Knock-Out, Standard Deviation, Two Tailed Test

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    Clone Assay:

    Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease
    Article Snippet: .. Etoposide was from Selleckchem (#S1225). pGW1-Httex1 -Q17 -GFP and pGW1-Httex1 -Q72 -GFP were described ( , ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHttex1 -Q46 was derived from pGW1-mHttex1 -Q46 -GFP plasmid ( ). .. Non-targeting siRNA (#D-001810) and siRNA against SK2 (#L-041258) were from Dharmacon.

    Irradiation:

    Article Title: Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies
    Article Snippet: .. For etoposide treatment, the cells were treated with 0.025 or 0.05 µg/ml etoposide (Selleck Chemicals, Houston, TX, USA) for 24 h. Next, the cells were irradiated as aforementioned and subsequently collected at different time points as indicated in the applicable figures for further analysis. ..

    Cell Culture:

    Article Title: Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53
    Article Snippet: .. PI and TMRM staining Primary dermal fibroblasts, primary cortical neurons, and HEK293 cells were cultured and treated with dimethyl sulfoxide, etoposide (Selleck, Houston, TX, USA), or cisplatin (Selleck). .. Cells were incubated with Hoechst (Sigma) and PI (Beyotime, Haimen, Jiangsu, China) for 5 min, washed with phosphate-buffered saline (PBS), and detected under a fluorescence microscope.

    Incubation:

    Article Title: Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer
    Article Snippet: .. H1694 cell viability assays NCI-H1694 with APC knockdown (shAPC#1, shAPC#2) and shAPC#2 cells overexpressing APC, and their appropriate controls (shScr for knockdown and GFP for overexpression experiments) were plated in 96 well plates in RPMI medium containing 10% FBS, with cisplatin obtained from Selleck chemicals (Houston, TX, USA), diluted in DMF, for 48 h and etoposide, diluted in DMSO (Selleck chemicals), for 72 h. Cell viability at the end of each incubation period was measured using alamarBlue (catalog number DAL1025) obtained from ThermoFisher (Waltham, MA, USA) according to manufacturer’s instructions. .. Briefly, cells were incubated with alamarBlue for 24 h, after which alamarBlue reduction was measured by fluorescence on a Spectramax M5 plate reader manufactured by Molecular Devices (Sunnyvale, CA, USA).

    other:

    Article Title: Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1
    Article Snippet: Drugs Dinaciclib, doxorubicin, etoposide, cytarabine, flavopiridol, SNS-032, and PHA-767491 were purchased from Selleck Chemicals (Houston, TX).

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor
    Article Snippet: PD0332991, Etoposide (VP-16), and cisplatin are from Selleckchem (Houston, TX); stock solutions re in DMSO except for cisplatin, which is in dimethylformamide.

    Plasmid Preparation:

    Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease
    Article Snippet: .. Etoposide was from Selleckchem (#S1225). pGW1-Httex1 -Q17 -GFP and pGW1-Httex1 -Q72 -GFP were described ( , ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHttex1 -Q46 was derived from pGW1-mHttex1 -Q46 -GFP plasmid ( ). .. Non-targeting siRNA (#D-001810) and siRNA against SK2 (#L-041258) were from Dharmacon.

    Staining:

    Article Title: Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription-independent function of p53
    Article Snippet: .. PI and TMRM staining Primary dermal fibroblasts, primary cortical neurons, and HEK293 cells were cultured and treated with dimethyl sulfoxide, etoposide (Selleck, Houston, TX, USA), or cisplatin (Selleck). .. Cells were incubated with Hoechst (Sigma) and PI (Beyotime, Haimen, Jiangsu, China) for 5 min, washed with phosphate-buffered saline (PBS), and detected under a fluorescence microscope.

    Over Expression:

    Article Title: Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer
    Article Snippet: .. H1694 cell viability assays NCI-H1694 with APC knockdown (shAPC#1, shAPC#2) and shAPC#2 cells overexpressing APC, and their appropriate controls (shScr for knockdown and GFP for overexpression experiments) were plated in 96 well plates in RPMI medium containing 10% FBS, with cisplatin obtained from Selleck chemicals (Houston, TX, USA), diluted in DMF, for 48 h and etoposide, diluted in DMSO (Selleck chemicals), for 72 h. Cell viability at the end of each incubation period was measured using alamarBlue (catalog number DAL1025) obtained from ThermoFisher (Waltham, MA, USA) according to manufacturer’s instructions. .. Briefly, cells were incubated with alamarBlue for 24 h, after which alamarBlue reduction was measured by fluorescence on a Spectramax M5 plate reader manufactured by Molecular Devices (Sunnyvale, CA, USA).

    Derivative Assay:

    Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease
    Article Snippet: .. Etoposide was from Selleckchem (#S1225). pGW1-Httex1 -Q17 -GFP and pGW1-Httex1 -Q72 -GFP were described ( , ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHttex1 -Q46 was derived from pGW1-mHttex1 -Q46 -GFP plasmid ( ). .. Non-targeting siRNA (#D-001810) and siRNA against SK2 (#L-041258) were from Dharmacon.

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    Selleck Chemicals etoposide
    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, <t>etoposide.</t> Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.
    Etoposide, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.

    Journal: Scientific Reports

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    doi: 10.1038/srep14391

    Figure Lengend Snippet: HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.

    Article Snippet: PD0332991, Etoposide (VP-16), and cisplatin are from Selleckchem (Houston, TX); stock solutions re in DMSO except for cisplatin, which is in dimethylformamide.

    Techniques:

    Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.

    Journal: Scientific Reports

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    doi: 10.1038/srep14391

    Figure Lengend Snippet: Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.

    Article Snippet: PD0332991, Etoposide (VP-16), and cisplatin are from Selleckchem (Houston, TX); stock solutions re in DMSO except for cisplatin, which is in dimethylformamide.

    Techniques: Single Cell Tracking

    ABT-199 synergizes strongly with lymphoma chemotherapy agents that affect MCL1 levels ( a ) Doxorubicin vs. dinaciclib activity comparison as in Figure 1a . ( b ) The indicated lines were treated with doxorubicin at the indicated times and concentrations and subjected to western blotting. ( c-e ) Viability and CI vs. Fa after 24 hours’ exposure to doxorubicin ( c ), etoposide ( d ), or cytarabine ( e ) alone or in combination with ABT-199 in Riva, U2932, and VavP- Bcl2/c-MYC murine tumor cells. Viability shown at 500 nM (500 ng/mL for doxorubicin; quadruplicates ± SEM.)

    Journal: Leukemia

    Article Title: Synergistic Induction of Apoptosis in High-Risk DLBCL by BCL2 Inhibition with ABT-199 Combined With Pharmacologic Loss of MCL1

    doi: 10.1038/leu.2015.99

    Figure Lengend Snippet: ABT-199 synergizes strongly with lymphoma chemotherapy agents that affect MCL1 levels ( a ) Doxorubicin vs. dinaciclib activity comparison as in Figure 1a . ( b ) The indicated lines were treated with doxorubicin at the indicated times and concentrations and subjected to western blotting. ( c-e ) Viability and CI vs. Fa after 24 hours’ exposure to doxorubicin ( c ), etoposide ( d ), or cytarabine ( e ) alone or in combination with ABT-199 in Riva, U2932, and VavP- Bcl2/c-MYC murine tumor cells. Viability shown at 500 nM (500 ng/mL for doxorubicin; quadruplicates ± SEM.)

    Article Snippet: Drugs Dinaciclib, doxorubicin, etoposide, cytarabine, flavopiridol, SNS-032, and PHA-767491 were purchased from Selleck Chemicals (Houston, TX).

    Techniques: Activity Assay, Western Blot

    Investigation of apoptotic and necrotic cell death mechanisms in UCCs induced by class I HDAC inhibitor 4SC-202. Relative cell viability measured by MTT-assay in VM-CUB1 and UM-UC-3 cells following 4SC-202 treatment (0.5/2.5 μM, 48 h) in combination with Q-VD-OPh (pan-caspase inhibitor) or Necrox-2 (necrosis inhibitor). Etoposide or H 2 O 2 treatments were used as positive controls. The determined significances of the treated cells relate to the DMSO solvent control. Additional significances between the different treatments are shown by brackets

    Journal: Targeted Oncology

    Article Title: Evaluation of the Therapeutic Potential of the Novel Isotype Specific HDAC Inhibitor 4SC-202 in Urothelial Carcinoma Cell Lines

    doi: 10.1007/s11523-016-0444-7

    Figure Lengend Snippet: Investigation of apoptotic and necrotic cell death mechanisms in UCCs induced by class I HDAC inhibitor 4SC-202. Relative cell viability measured by MTT-assay in VM-CUB1 and UM-UC-3 cells following 4SC-202 treatment (0.5/2.5 μM, 48 h) in combination with Q-VD-OPh (pan-caspase inhibitor) or Necrox-2 (necrosis inhibitor). Etoposide or H 2 O 2 treatments were used as positive controls. The determined significances of the treated cells relate to the DMSO solvent control. Additional significances between the different treatments are shown by brackets

    Article Snippet: For distinguishing modes of cell death, cells were treated additionally with Necrox-2 (20 μM, sc-391057, Santa Cruz, Dallas, USA) or Q-VD-OPh (30 μM, SML0063, Sigma Aldrich); Etoposide was used to induce apoptosis (25 μM, S1225, Selleck Chemicals) and H2 O2 to induce necrosis (30 μM, 107210, Merck, Darmstadt, Germany) as previously described [ ].

    Techniques: MTT Assay

    Effects of triple combination treatments of vorinostat with cisplatin and etoposide on the viability and apoptosis of SCLC cells. H209 and H146 cells were treated with or without vorinostat in combination with cisplatin ( a vorinostat at 0.8 μM, and cisplatin and etoposide both at 1 μM; b vorinostat at 0.4 μM, cisplatin at 0.2 μM, and etoposide at 0.6 μM) for 24 h. Cell viability was determined using the MTS assay, and data were represented as mean ± SD in triplicate. A significant reduction in cell viability was documented (*, P

    Journal: BMC Cancer

    Article Title: Vorinostat enhances the cisplatin-mediated anticancer effects in small cell lung cancer cells

    doi: 10.1186/s12885-016-2888-7

    Figure Lengend Snippet: Effects of triple combination treatments of vorinostat with cisplatin and etoposide on the viability and apoptosis of SCLC cells. H209 and H146 cells were treated with or without vorinostat in combination with cisplatin ( a vorinostat at 0.8 μM, and cisplatin and etoposide both at 1 μM; b vorinostat at 0.4 μM, cisplatin at 0.2 μM, and etoposide at 0.6 μM) for 24 h. Cell viability was determined using the MTS assay, and data were represented as mean ± SD in triplicate. A significant reduction in cell viability was documented (*, P

    Article Snippet: Materials Vorinostat (purity > 99 %) was purchased from AbMole BioScience (Houston, TX, USA); cisplatin (purity > 99 %) and etoposide (purity > 99 %) were purchased from Selleck Chemicals (Houston, TX, USA).

    Techniques: MTS Assay