etoposide  (Millipore)


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  • 99
    Name:
    4 Maleimidobutyramidomethyl polystyrene
    Description:

    Catalog Number:
    63178
    Price:
    None
    Applications:
    Polymer(polystyrene)-supported maleimide ((4-Maleimidobutyramidomethyl)polystyrene)) may be use for solid phase trapping of thiols from solutions and for the immobilization of sulhydryl-containing substances such as proteins and peptides.
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    Structured Review

    Millipore etoposide
    4 Maleimidobutyramidomethyl polystyrene

    https://www.bioz.com/result/etoposide/product/Millipore
    Average 99 stars, based on 474 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis"

    Article Title: Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis

    Journal: Chinese Medical Journal

    doi: 10.1097/CM9.0000000000000553

    Validation of iTRAQ data through Western blot analysis. H2AX was selected for validation of the expression alteration trends through Western blot analysis. The results showed that H2AX is downregulated in Caki-1 cells compared with XIAP knockdown cells after etoposide treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.
    Figure Legend Snippet: Validation of iTRAQ data through Western blot analysis. H2AX was selected for validation of the expression alteration trends through Western blot analysis. The results showed that H2AX is downregulated in Caki-1 cells compared with XIAP knockdown cells after etoposide treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.

    Techniques Used: Western Blot, Expressing

    2) Product Images from "Inhibition of Apoptosis in Chlamydia-infected Cells: Blockade of Mitochondrial Cytochrome c Release and Caspase Activation "

    Article Title: Inhibition of Apoptosis in Chlamydia-infected Cells: Blockade of Mitochondrial Cytochrome c Release and Caspase Activation

    Journal: The Journal of Experimental Medicine

    doi:

    ( A ) Chlamydia-infected HeLa cells are resistant to apoptosis induced by staurosporine. HeLa cells with ( c–e and h–j ) or without ( a , b , f , and g ) chlamydial infection at an MOI of 5 (+ high , top , c , d , h , and i ) or 0.5 (+ low , e and j ) were treated with ( b , d , e , g , i , and j ) or without ( a , c , f , and h ) 1 μM staurosporine for 4 h. The cell samples were then either stained with Hoechst dye and viewed under a fluorescence microscope ( a–e ) or doubly labeled with dUTP ( green ) and an antichlamydial antibody ( red ) and viewed under a confocal microscope ( f–j ) as described in Materials and Methods. Hoechst dye stained both HeLa cell nuclei ( black arrowhead , apoptotic nuclei) and the cytoplasmic chlamydial inclusion bodies ( white arrowheads ). ( B ) Chlamydia-infected HeLa cells are resistant to apoptosis induced by multiple apoptotic stimuli. Host cells with ( solid bars ) or without ( open bars ) chlamydial infection were stimulated with staurosporine ( Stau ), etoposide ( ET ), granzyme B/perforin ( GB / P ), an anti-Fas IgM antibody CH11 ( αFas ), or TNF-α. The cell samples were then stained with Hoechst dye. Cells from five random fields were counted under an object lens of ×40 and the percent of apoptotic cells were calculated (displayed along the y-axis). The figure shows the results from three independent experiments. The variations between the three experiments were
    Figure Legend Snippet: ( A ) Chlamydia-infected HeLa cells are resistant to apoptosis induced by staurosporine. HeLa cells with ( c–e and h–j ) or without ( a , b , f , and g ) chlamydial infection at an MOI of 5 (+ high , top , c , d , h , and i ) or 0.5 (+ low , e and j ) were treated with ( b , d , e , g , i , and j ) or without ( a , c , f , and h ) 1 μM staurosporine for 4 h. The cell samples were then either stained with Hoechst dye and viewed under a fluorescence microscope ( a–e ) or doubly labeled with dUTP ( green ) and an antichlamydial antibody ( red ) and viewed under a confocal microscope ( f–j ) as described in Materials and Methods. Hoechst dye stained both HeLa cell nuclei ( black arrowhead , apoptotic nuclei) and the cytoplasmic chlamydial inclusion bodies ( white arrowheads ). ( B ) Chlamydia-infected HeLa cells are resistant to apoptosis induced by multiple apoptotic stimuli. Host cells with ( solid bars ) or without ( open bars ) chlamydial infection were stimulated with staurosporine ( Stau ), etoposide ( ET ), granzyme B/perforin ( GB / P ), an anti-Fas IgM antibody CH11 ( αFas ), or TNF-α. The cell samples were then stained with Hoechst dye. Cells from five random fields were counted under an object lens of ×40 and the percent of apoptotic cells were calculated (displayed along the y-axis). The figure shows the results from three independent experiments. The variations between the three experiments were

    Techniques Used: Infection, Staining, Fluorescence, Microscopy, Labeling

    3) Product Images from "ATM/Wip1 activities at chromatin control Plk1 re‐activation to determine G2 checkpoint duration"

    Article Title: ATM/Wip1 activities at chromatin control Plk1 re‐activation to determine G2 checkpoint duration

    Journal: The EMBO Journal

    doi: 10.15252/embj.201696082

    A FRET‐based biosensor to monitor the activity of ATM/ATR kinase in live cells Acceptor photobleaching of H2B‐ATKAR. U2OS cells expressing H2B‐ATKAR were photobleached using a 514 nm laser, and images were acquired by using CFP‐YFP, YFP‐YFP, and CFP‐CFP excitation‐emission before and after photobleaching. The bleached area is visible in the YFP‐YFP images. Kinetics of H2B‐ATKAR 1/FRET change after treatment with etoposide (Eto), neocarzinostatin (NCS) or cytolethal distending toxin (CDT). Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing H2B‐ATKAR. Graph shows average and SD of at least 15 cells. Time point 0 indicates addition of drugs. Scale bar: 15 μm. Heat map indicates 1/FRET (AU). Kinetics of ATKAR 1/FRET change after treatment with etoposide (Eto), neocarzinostatin (NCS), or cytolethal distending toxin (CDT). Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing H2B‐ATKAR. Graph shows average and SD of at least 15 cells. Time point 0 indicates addition of drugs. Scale bar: 15 μm. Heat map indicates 1/FRET (AU). H2B‐ATKAR phosphorylation after NCS addition depends on ATM. GFP pull‐down from U2OS cells expressing H2B‐ATKAR treated with NCS (5 nM) or exposed to IR (5 Gy) or UVC (10 J/m 2 ). Immunoblots were probed with the indicated antibodies. Change in ATKAR FRET‐ratio after NCS addition depends on ATM. Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing ATKAR. Time point 0 indicates addition of 5 nM NCS, and time point 1 indicates addition of KU60019 (10 μM, ATMi), VE821 (1 μM, ATRi), or NU7026 (10 μM, DNAPKi). Graph shows average and SD of at least 7 cells. Scale bar: 10 μm. Heat map indicates 1/FRET (AU). GFP pull‐down from U2OS cells expressing ATKAR wild‐type (Wt) or alanine mutant (Ala) treated with NCS (10 nM) in presence or absence of ATM inhibitor. Immunoblot analysis shows the phosphorylation of ATKAR is on the expected target site residue and is ATM‐dependent upon NCS treatment. H2B‐ATKAR phosphorylation after NCS treatment depends on ATM. GFP pull‐down from AT cells expressing H2B‐PLK1 or H2B‐ATKAR FRET probe treated with 5–10 nM NCS or 20–40 J/m 2 UV‐C. Immunoblots were probed with indicated antibodies. Note that phosphorylation of H2B‐ATKAR is not induced by DNA damage in cells lacking ATM. Quantification of 1/FRET of MCF‐7 and RPE cells expressing H2B‐ATKAR treated with the indicated concentrations of NCS. Graph shows average of at least 10 cells per condition. ATKAR phosphorylation depends on ATM activity long after NCS addition. U2OS cells expressing ATKAR were treated with 5 nM NCS. The indicated inhibitors were added 24 h later. Graph shows average and SD of at least 15 cells. Similar dynamics of phosphorylation of Plk1 sensor on chromatin or nucleoplasm. Quantification of nuclear 1/FRET of U2OS cells expressing either H2B‐Plk1 or Plk1 biosensor. Cells are synchronized upon mitotic entry in silico .
    Figure Legend Snippet: A FRET‐based biosensor to monitor the activity of ATM/ATR kinase in live cells Acceptor photobleaching of H2B‐ATKAR. U2OS cells expressing H2B‐ATKAR were photobleached using a 514 nm laser, and images were acquired by using CFP‐YFP, YFP‐YFP, and CFP‐CFP excitation‐emission before and after photobleaching. The bleached area is visible in the YFP‐YFP images. Kinetics of H2B‐ATKAR 1/FRET change after treatment with etoposide (Eto), neocarzinostatin (NCS) or cytolethal distending toxin (CDT). Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing H2B‐ATKAR. Graph shows average and SD of at least 15 cells. Time point 0 indicates addition of drugs. Scale bar: 15 μm. Heat map indicates 1/FRET (AU). Kinetics of ATKAR 1/FRET change after treatment with etoposide (Eto), neocarzinostatin (NCS), or cytolethal distending toxin (CDT). Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing H2B‐ATKAR. Graph shows average and SD of at least 15 cells. Time point 0 indicates addition of drugs. Scale bar: 15 μm. Heat map indicates 1/FRET (AU). H2B‐ATKAR phosphorylation after NCS addition depends on ATM. GFP pull‐down from U2OS cells expressing H2B‐ATKAR treated with NCS (5 nM) or exposed to IR (5 Gy) or UVC (10 J/m 2 ). Immunoblots were probed with the indicated antibodies. Change in ATKAR FRET‐ratio after NCS addition depends on ATM. Time‐lapse sequence (left) or quantification of 1/FRET (right) of U2OS cells expressing ATKAR. Time point 0 indicates addition of 5 nM NCS, and time point 1 indicates addition of KU60019 (10 μM, ATMi), VE821 (1 μM, ATRi), or NU7026 (10 μM, DNAPKi). Graph shows average and SD of at least 7 cells. Scale bar: 10 μm. Heat map indicates 1/FRET (AU). GFP pull‐down from U2OS cells expressing ATKAR wild‐type (Wt) or alanine mutant (Ala) treated with NCS (10 nM) in presence or absence of ATM inhibitor. Immunoblot analysis shows the phosphorylation of ATKAR is on the expected target site residue and is ATM‐dependent upon NCS treatment. H2B‐ATKAR phosphorylation after NCS treatment depends on ATM. GFP pull‐down from AT cells expressing H2B‐PLK1 or H2B‐ATKAR FRET probe treated with 5–10 nM NCS or 20–40 J/m 2 UV‐C. Immunoblots were probed with indicated antibodies. Note that phosphorylation of H2B‐ATKAR is not induced by DNA damage in cells lacking ATM. Quantification of 1/FRET of MCF‐7 and RPE cells expressing H2B‐ATKAR treated with the indicated concentrations of NCS. Graph shows average of at least 10 cells per condition. ATKAR phosphorylation depends on ATM activity long after NCS addition. U2OS cells expressing ATKAR were treated with 5 nM NCS. The indicated inhibitors were added 24 h later. Graph shows average and SD of at least 15 cells. Similar dynamics of phosphorylation of Plk1 sensor on chromatin or nucleoplasm. Quantification of nuclear 1/FRET of U2OS cells expressing either H2B‐Plk1 or Plk1 biosensor. Cells are synchronized upon mitotic entry in silico .

    Techniques Used: Activity Assay, Expressing, Sequencing, Western Blot, Mutagenesis, In Silico

    4) Product Images from "Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner"

    Article Title: Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner

    Journal: bioRxiv

    doi: 10.1101/614040

    Genetic dependencies of the IR-stimulated random integration ( A ) Colony-based S-RI assay performed with mES cells deficient for key DSB repair and DNA damage response (DDR) proteins. The numbers of colonies obtained after puromycin selection were normalized to the unirradiated control. Means from at least 3 independent experiments, fitted with sigmoid curve, are plotted; error bars show s.e.m. ( B ) mES lines deficient for end joining proteins were assayed as in panel (A). ( C ) H2ax −/− (N) cells were complemented with versions of H2AX containing mutations in the residues involved in key post-translational modifications during the DNA damage response. A single copy of the respective H2AX genes was inserted in to the Rosa26 locus. n=3, s.e.m. K4R designates a mutant in which four lysines subject to ubiquitination were replaced with arginines. ( D ) S-RI response to broad irradiation dose range of H2ax −/− and wild-type mES cells was measured in a colony-based S-RI assay. Each point represents means of at least six biological replicas, adjusted for reduced survival ( [ Fig S2D ] ), error bars indicated s.e.m. Both (N) and (A) H2ax −/-/ lines were used. ( E ) RI stimulation by etoposide measured by colony formation S-RI assay. Experiment was performed twice, with two independent H2ax -/-/ lines in each ((A) and (N)), data for each genotype was averaged, error bars indicate s.e.m.
    Figure Legend Snippet: Genetic dependencies of the IR-stimulated random integration ( A ) Colony-based S-RI assay performed with mES cells deficient for key DSB repair and DNA damage response (DDR) proteins. The numbers of colonies obtained after puromycin selection were normalized to the unirradiated control. Means from at least 3 independent experiments, fitted with sigmoid curve, are plotted; error bars show s.e.m. ( B ) mES lines deficient for end joining proteins were assayed as in panel (A). ( C ) H2ax −/− (N) cells were complemented with versions of H2AX containing mutations in the residues involved in key post-translational modifications during the DNA damage response. A single copy of the respective H2AX genes was inserted in to the Rosa26 locus. n=3, s.e.m. K4R designates a mutant in which four lysines subject to ubiquitination were replaced with arginines. ( D ) S-RI response to broad irradiation dose range of H2ax −/− and wild-type mES cells was measured in a colony-based S-RI assay. Each point represents means of at least six biological replicas, adjusted for reduced survival ( [ Fig S2D ] ), error bars indicated s.e.m. Both (N) and (A) H2ax −/-/ lines were used. ( E ) RI stimulation by etoposide measured by colony formation S-RI assay. Experiment was performed twice, with two independent H2ax -/-/ lines in each ((A) and (N)), data for each genotype was averaged, error bars indicate s.e.m.

    Techniques Used: Selection, Mutagenesis, Irradiation

    5) Product Images from "The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival"

    Article Title: The DRY Box and C-Terminal Domain of the Human Cytomegalovirus US27 Gene Product Play a Role in Promoting Cell Growth and Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113427

    HEK293 cells expressing wild type US27 are more resistant to apoptosis. Cells were treated with 10 µM etoposide to induce apoptosis. A) After 48 hours, cells were stained with propidium iodide and Annexin V then analyzed by flow cytometry. B) The average percent positive cells for Annexin V, propidium iodide (PI) or both (double positive, DP) from three independent experiments, as indicated in A above. * indicates p
    Figure Legend Snippet: HEK293 cells expressing wild type US27 are more resistant to apoptosis. Cells were treated with 10 µM etoposide to induce apoptosis. A) After 48 hours, cells were stained with propidium iodide and Annexin V then analyzed by flow cytometry. B) The average percent positive cells for Annexin V, propidium iodide (PI) or both (double positive, DP) from three independent experiments, as indicated in A above. * indicates p

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

    6) Product Images from "A cell-penetrating antibody inhibits human RAD51 via direct binding"

    Article Title: A cell-penetrating antibody inhibits human RAD51 via direct binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx871

    3E10 inhibits RAD51 nuclear localization and foci formation after treatment with etoposide. ( A ) U2OS cells were left untreated or pretreated with purified 2xMBP-3E10 scFv proteins and then treated with etoposide and fixed for immunofluorescence. ( B ) Cells with 10 or more RAD51 foci were scored for each condition. ( C ) Representative images of treated U2OS cells. ( D ) The cytoplasmic fraction of RAD51 was also scored for each condition. Error bars represent the SEM; *** P
    Figure Legend Snippet: 3E10 inhibits RAD51 nuclear localization and foci formation after treatment with etoposide. ( A ) U2OS cells were left untreated or pretreated with purified 2xMBP-3E10 scFv proteins and then treated with etoposide and fixed for immunofluorescence. ( B ) Cells with 10 or more RAD51 foci were scored for each condition. ( C ) Representative images of treated U2OS cells. ( D ) The cytoplasmic fraction of RAD51 was also scored for each condition. Error bars represent the SEM; *** P

    Techniques Used: Purification, Immunofluorescence

    7) Product Images from "SPOP is essential for DNA–protein cross-link repair in prostate cancer cells: SPOP-dependent removal of topoisomerase 2A from the topoisomerase 2A-DNA cleavage complex"

    Article Title: SPOP is essential for DNA–protein cross-link repair in prostate cancer cells: SPOP-dependent removal of topoisomerase 2A from the topoisomerase 2A-DNA cleavage complex

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E19-08-0456

    Increased level of γH2AX by depletion of SPOP was not enhanced by topoisomerase inhibitors and hydroxyurea. (A) Western blots of cell lysates prepared from control or SPOP-knockdown C4-2 cells incubated with inhibitors of DNA repair. Cells were treated with irinotecan (Irino), hydroxyurea (HU), or etoposide (Etop) at the indicated concentrations in 10% FBS–containing medium for 24 h. (B) Quantitation of A. The ratio of γH2AX/H2AX was analyzed from three independent experiments. Data show the mean ± SEM. *, p
    Figure Legend Snippet: Increased level of γH2AX by depletion of SPOP was not enhanced by topoisomerase inhibitors and hydroxyurea. (A) Western blots of cell lysates prepared from control or SPOP-knockdown C4-2 cells incubated with inhibitors of DNA repair. Cells were treated with irinotecan (Irino), hydroxyurea (HU), or etoposide (Etop) at the indicated concentrations in 10% FBS–containing medium for 24 h. (B) Quantitation of A. The ratio of γH2AX/H2AX was analyzed from three independent experiments. Data show the mean ± SEM. *, p

    Techniques Used: Western Blot, Incubation, Quantitation Assay

    Sensitivity to etoposide in SPOP-depleted and SPOP-overexpressing C4-2 cells. (A) Western blots of cell lysates prepared from control or SPOP-knockdown C4-2 cells incubated with 50 µM etoposide (Etop) for the indicated time in 10% FBS–containing medium. (B) Quantitation of A. Ratio of γH2AX/H2AX was analyzed from three independent experiments. Data show the mean ± SEM. *, p
    Figure Legend Snippet: Sensitivity to etoposide in SPOP-depleted and SPOP-overexpressing C4-2 cells. (A) Western blots of cell lysates prepared from control or SPOP-knockdown C4-2 cells incubated with 50 µM etoposide (Etop) for the indicated time in 10% FBS–containing medium. (B) Quantitation of A. Ratio of γH2AX/H2AX was analyzed from three independent experiments. Data show the mean ± SEM. *, p

    Techniques Used: Western Blot, Incubation, Quantitation Assay

    8) Product Images from "Targeting Glutathione S-transferase M4 in Ewing sarcoma"

    Article Title: Targeting Glutathione S-transferase M4 in Ewing sarcoma

    Journal: Frontiers in Pediatrics

    doi: 10.3389/fped.2014.00083

    The GST inhibitor NBDHEX inhibits Ewing sarcoma cell proliferation and oncogenic transformation and increases the efficacy of etoposide . (A) , NBDHEX inhibits Ewing sarcoma cell growth in culture. Ewing sarcoma TC71 and A673 cells, rhabdomyosarcoma RH30 cells, and HEK293 cells were subjected to treatment with the indicated concentrations of NBDHEX. Cell proliferation was assessed by MTT assays. The experiment was repeated three times and results of one representative repeat are shown as means ± SD of technical triplicate. (B) NBDHEX inhibits colony formation of Ewing sarcoma cells in soft agar. TC71 and A673 cells were treated with NBDHEX at the indicated concentrations, and then were seeded in soft agar to assess anchorage-independent growth. The experiment was repeated three times and results of one representative repeat are shown as means ± SD of technical duplicate. Asterisk indicates p
    Figure Legend Snippet: The GST inhibitor NBDHEX inhibits Ewing sarcoma cell proliferation and oncogenic transformation and increases the efficacy of etoposide . (A) , NBDHEX inhibits Ewing sarcoma cell growth in culture. Ewing sarcoma TC71 and A673 cells, rhabdomyosarcoma RH30 cells, and HEK293 cells were subjected to treatment with the indicated concentrations of NBDHEX. Cell proliferation was assessed by MTT assays. The experiment was repeated three times and results of one representative repeat are shown as means ± SD of technical triplicate. (B) NBDHEX inhibits colony formation of Ewing sarcoma cells in soft agar. TC71 and A673 cells were treated with NBDHEX at the indicated concentrations, and then were seeded in soft agar to assess anchorage-independent growth. The experiment was repeated three times and results of one representative repeat are shown as means ± SD of technical duplicate. Asterisk indicates p

    Techniques Used: Transformation Assay, MTT Assay

    GSTM4 inhibits etoposide-mediated JNK activation and apoptosis by interacting with ASK1 . (A) Decreasing GSTM4 levels increases JNK activation induced by etoposide. Control- (Luc-RNAi) and GSTM4-silenced (GSTM4-4-RNAi and GSTM4-5 RNAi) cells were treated with or without etoposide (20 μg/ml) for 3 h. Cell lysates were subjected to JNK immunoprecipitation and JNK kinase assays using c-jun as the substrate. The result shown is representative of three experimental repeats. (B) Decreasing GSTM4 levels increases apoptosis induced by etoposide. Control-silenced (Luc-RNAi) and GSTM4-silenced (GSTM4-4-RNAi and GSTM4-5 RNAi) cells were treated with or without etoposide, as in (A) above. Expression levels of GSTM4, BAD, BAX, and CASP3 then were evaluated by qRT-PCR analyses. “ns” indicates not significant, * p
    Figure Legend Snippet: GSTM4 inhibits etoposide-mediated JNK activation and apoptosis by interacting with ASK1 . (A) Decreasing GSTM4 levels increases JNK activation induced by etoposide. Control- (Luc-RNAi) and GSTM4-silenced (GSTM4-4-RNAi and GSTM4-5 RNAi) cells were treated with or without etoposide (20 μg/ml) for 3 h. Cell lysates were subjected to JNK immunoprecipitation and JNK kinase assays using c-jun as the substrate. The result shown is representative of three experimental repeats. (B) Decreasing GSTM4 levels increases apoptosis induced by etoposide. Control-silenced (Luc-RNAi) and GSTM4-silenced (GSTM4-4-RNAi and GSTM4-5 RNAi) cells were treated with or without etoposide, as in (A) above. Expression levels of GSTM4, BAD, BAX, and CASP3 then were evaluated by qRT-PCR analyses. “ns” indicates not significant, * p

    Techniques Used: Activation Assay, Immunoprecipitation, Expressing, Quantitative RT-PCR

    9) Product Images from "Egr-1 regulates the transcription of the BRCA1 gene by etoposide"

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2013.46.2.202

    Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).
    Figure Legend Snippet: Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

    Techniques Used: Expressing, Isolation, Northern Blot, Labeling, Western Blot, Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay

    Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.
    Figure Legend Snippet: Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Purification, Recombinant, Incubation, Labeling

    Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.
    Figure Legend Snippet: Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.

    Techniques Used: Expressing, Western Blot, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    10) Product Images from "Paclitaxel and etoposide co-loaded polymeric nanoparticles for the effective combination therapy against human osteosarcoma"

    Article Title: Paclitaxel and etoposide co-loaded polymeric nanoparticles for the effective combination therapy against human osteosarcoma

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-015-0086-4

    Schematic illustration. (A) Schematic representation of assembly of PLGA polymers and paclitaxel and etoposide towards the formation of drug-loaded nanoparticles. Schematic illustration of self-assembling process of polymeric nanoparticles. (B) Typical size distribution analysis of PLGA NP by dynamic light scattering technique (C) transmission electron microscope (TEM) imaging of drug-loaded PLGA NP.
    Figure Legend Snippet: Schematic illustration. (A) Schematic representation of assembly of PLGA polymers and paclitaxel and etoposide towards the formation of drug-loaded nanoparticles. Schematic illustration of self-assembling process of polymeric nanoparticles. (B) Typical size distribution analysis of PLGA NP by dynamic light scattering technique (C) transmission electron microscope (TEM) imaging of drug-loaded PLGA NP.

    Techniques Used: Transmission Assay, Microscopy, Transmission Electron Microscopy, Imaging

    11) Product Images from "Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3 *"

    Article Title: Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3 *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M110.006007

    Palmitoylation-deficient mutant Plscr3 confers reduced apoptotic function. A , RAW 264.7 macrophages stably expressing EV, wt Plscr3, or 5A mutant Plscr3 were treated with vehicle (DMSO) or different concentrations of etoposide (Etop) for 24 h. Cells were
    Figure Legend Snippet: Palmitoylation-deficient mutant Plscr3 confers reduced apoptotic function. A , RAW 264.7 macrophages stably expressing EV, wt Plscr3, or 5A mutant Plscr3 were treated with vehicle (DMSO) or different concentrations of etoposide (Etop) for 24 h. Cells were

    Techniques Used: Mutagenesis, Stable Transfection, Expressing

    12) Product Images from "Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation"

    Article Title: Transcription-coupled nucleotide excision repair is coordinated by ubiquitin and SUMO in response to ultraviolet irradiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz977

    UV-induced SUMOylation of CSB is dependent on transcription and located at the N-terminus. ( A ) U2OS cells stably expressing His10-SUMO2 were left untreated or were treated with either DMSO, or 0.02% MMS, 50 μM etoposide, 2 mM hydroxyurea, 4 Gy IR or 20 J/m 2 UV. Times indicate treatment period of cells with the compound before lysis. For IR and UV treated cells, times represent recovery period after treatment. SUMO2 conjugates were enriched by Ni-NTA pulldown (PD). Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB or SUMO2/3. MMS, methyl methanesulfonate; IR, ionizing radiation; UV, ultraviolet light irradiation. ( B ) U2OS cells stably expressing His10-SUMO2 were treated with DRB or α-amanitin and/or UV irradiation (20 J/m 2 ). Times indicate treatment period with the compound before lysis. For irradiation, these times indicate recovery period after irradiation prior to lysis. For the DRB and UV irradiated sample, cells were treated with DRB 3 h prior to UV irradiation and lysed 30 min after the UV treatment. For α-amanitin and UV irradiated samples, cells were treated 24 h prior to UV irradiation and lysed 30 min after UV treatment. SUMO2 conjugates were enriched by Ni-NTA pulldown. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB, p-RPB1 (S2/S5), RPB1, or SUMO2/3. DRB, 5,6-dichlorobenzimidazole 1-β- d -ribofuranoside. ( C ) Schematic overview of CSB including known domains and localization of SUMOylation consensus sites (ψKXE). ( D ) U2OS cells stably expressing Flag-SUMO2 were infected with retroviruses encoding different SUMOylation consensus site mutants of GFP-CSB, as indicated. Cells were treated with UV irradiation (20 J/m 2 ) and lysed after 1 h recovery. SUMO2 conjugates were enriched by Flag IP. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against GFP or SUMO2/3. (E) CS1AN cells stably expressing His10-SUMO2 were infected with lentiviruses encoding different CSB SUMOylation consensus site mutants. Cells were treated with UV irradiation (20 J/m 2 ) and lysed after 30 min recovery. SUMO2 conjugates were enriched by Ni-NTA pulldown. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB or SUMO2/3. * marks the exogenously expressed CSB construct. ** marks the CSB-piggyBac transposable element derived three fusion (CPFP) ( 57 ). K32, 205R (2KR); K32, 205, 481, 1359, 1489R (5KR). (F) Quantification of (E). Relative amount of SUMOylated CSB was determined based on immunoblots. Intensities were corrected for exogenous CSB expression levels (see * in E) and protein loading as determined by expression of CPFP (** in E) and Ponceau S stain. Values were normalized to CSB WT SUMOylation. Error bars represent SD of the mean obtained from three independent experiments * P -value
    Figure Legend Snippet: UV-induced SUMOylation of CSB is dependent on transcription and located at the N-terminus. ( A ) U2OS cells stably expressing His10-SUMO2 were left untreated or were treated with either DMSO, or 0.02% MMS, 50 μM etoposide, 2 mM hydroxyurea, 4 Gy IR or 20 J/m 2 UV. Times indicate treatment period of cells with the compound before lysis. For IR and UV treated cells, times represent recovery period after treatment. SUMO2 conjugates were enriched by Ni-NTA pulldown (PD). Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB or SUMO2/3. MMS, methyl methanesulfonate; IR, ionizing radiation; UV, ultraviolet light irradiation. ( B ) U2OS cells stably expressing His10-SUMO2 were treated with DRB or α-amanitin and/or UV irradiation (20 J/m 2 ). Times indicate treatment period with the compound before lysis. For irradiation, these times indicate recovery period after irradiation prior to lysis. For the DRB and UV irradiated sample, cells were treated with DRB 3 h prior to UV irradiation and lysed 30 min after the UV treatment. For α-amanitin and UV irradiated samples, cells were treated 24 h prior to UV irradiation and lysed 30 min after UV treatment. SUMO2 conjugates were enriched by Ni-NTA pulldown. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB, p-RPB1 (S2/S5), RPB1, or SUMO2/3. DRB, 5,6-dichlorobenzimidazole 1-β- d -ribofuranoside. ( C ) Schematic overview of CSB including known domains and localization of SUMOylation consensus sites (ψKXE). ( D ) U2OS cells stably expressing Flag-SUMO2 were infected with retroviruses encoding different SUMOylation consensus site mutants of GFP-CSB, as indicated. Cells were treated with UV irradiation (20 J/m 2 ) and lysed after 1 h recovery. SUMO2 conjugates were enriched by Flag IP. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against GFP or SUMO2/3. (E) CS1AN cells stably expressing His10-SUMO2 were infected with lentiviruses encoding different CSB SUMOylation consensus site mutants. Cells were treated with UV irradiation (20 J/m 2 ) and lysed after 30 min recovery. SUMO2 conjugates were enriched by Ni-NTA pulldown. Total lysates and SUMO2-enriched fractions were analysed by immunoblotting using antibodies against CSB or SUMO2/3. * marks the exogenously expressed CSB construct. ** marks the CSB-piggyBac transposable element derived three fusion (CPFP) ( 57 ). K32, 205R (2KR); K32, 205, 481, 1359, 1489R (5KR). (F) Quantification of (E). Relative amount of SUMOylated CSB was determined based on immunoblots. Intensities were corrected for exogenous CSB expression levels (see * in E) and protein loading as determined by expression of CPFP (** in E) and Ponceau S stain. Values were normalized to CSB WT SUMOylation. Error bars represent SD of the mean obtained from three independent experiments * P -value

    Techniques Used: Stable Transfection, Expressing, Lysis, Irradiation, Infection, Construct, Derivative Assay, Western Blot, Staining

    SUMO and ubiquitin cooperate during TC-NER. The processing RNAPII is stalled upon encountering a DNA photolesion with helix distorting property, which is introduced by UV light, MMS or etoposide within an actively transcribed DNA region. In response to the stalled RNA polymerase, CSB is SUMOylated, recruited and stabilized at the lesion site. CSA is subsequently recruited to the site of damage and stimulates ubiquitination of RNAPII directly or indirectly. After a short recovery upon DNA damage, SUMOylated CSB is destabilized by the presence of CSA. The proteasomal degradation of ubiquitinated RNAPII is observed at a late stage (6 h) after UV irradiation, possibly related to failure of repair.
    Figure Legend Snippet: SUMO and ubiquitin cooperate during TC-NER. The processing RNAPII is stalled upon encountering a DNA photolesion with helix distorting property, which is introduced by UV light, MMS or etoposide within an actively transcribed DNA region. In response to the stalled RNA polymerase, CSB is SUMOylated, recruited and stabilized at the lesion site. CSA is subsequently recruited to the site of damage and stimulates ubiquitination of RNAPII directly or indirectly. After a short recovery upon DNA damage, SUMOylated CSB is destabilized by the presence of CSA. The proteasomal degradation of ubiquitinated RNAPII is observed at a late stage (6 h) after UV irradiation, possibly related to failure of repair.

    Techniques Used: Irradiation

    13) Product Images from "Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair"

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    Journal: Cell Research

    doi: 10.1038/s41422-018-0048-0

    H1.2 PARylation permits its displacement from chromatin upon DNA damage. a HeLa cells were transfected with GFP-H1.2 and treated with 20 μM Ku55933 or 2 μM Ku57788 for 4 h or 5 μM PJ34 for 1 h followed by laser micro-irradiation. Images were taken every 10 s for 5 min and quantifications of the IR path signal intensity were shown and ~15 IR paths from 10 separate cells were calculated. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for the indicated time. Chromatin was fractionated and analyzed by immunoblotting. c Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and chromatin was fractionated and analyzed by immunoblotting. d HeLa cells were transfected with FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h. Cell extracts were immunoprecipitated with FLAG-conjugated M2 beads. e Recombinant HIS-H1.2 was subjected to in vitro PARylation assay in the presence of NAD + or 10 μM PJ34, as indicated. f HeLa cells were transfected with wild-type or S188A mutated FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h, as indicated. Cells were extracted and immunoprecipitated with FLAG-conjugated M2 beads. g Recombinant wild-type, S188A mutated or C1-deleted (ΔC1) HIS-H1.2 were subjected to in vitro PARylation assay. h HeLa cells were transfected with wild-type, ΔC1 or S188A mutated GFP-H1.2 and subjected to laser micro-irradiation. Images were taken every 20 s for 5 min and representative images were shown. Quantifications were calculated as in a . The data represent the mean ± SD. Scale bars, 10 μm
    Figure Legend Snippet: H1.2 PARylation permits its displacement from chromatin upon DNA damage. a HeLa cells were transfected with GFP-H1.2 and treated with 20 μM Ku55933 or 2 μM Ku57788 for 4 h or 5 μM PJ34 for 1 h followed by laser micro-irradiation. Images were taken every 10 s for 5 min and quantifications of the IR path signal intensity were shown and ~15 IR paths from 10 separate cells were calculated. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for the indicated time. Chromatin was fractionated and analyzed by immunoblotting. c Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and chromatin was fractionated and analyzed by immunoblotting. d HeLa cells were transfected with FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h. Cell extracts were immunoprecipitated with FLAG-conjugated M2 beads. e Recombinant HIS-H1.2 was subjected to in vitro PARylation assay in the presence of NAD + or 10 μM PJ34, as indicated. f HeLa cells were transfected with wild-type or S188A mutated FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h, as indicated. Cells were extracted and immunoprecipitated with FLAG-conjugated M2 beads. g Recombinant wild-type, S188A mutated or C1-deleted (ΔC1) HIS-H1.2 were subjected to in vitro PARylation assay. h HeLa cells were transfected with wild-type, ΔC1 or S188A mutated GFP-H1.2 and subjected to laser micro-irradiation. Images were taken every 20 s for 5 min and representative images were shown. Quantifications were calculated as in a . The data represent the mean ± SD. Scale bars, 10 μm

    Techniques Used: Transfection, Irradiation, Immunoprecipitation, Recombinant, In Vitro

    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads
    Figure Legend Snippet: PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

    Techniques Used: Activation Assay, Clone Assay, Transfection, Fluorescence, Recombinant, Incubation, In Vitro, Phosphorylation Assay, GST Pulldown Assay, Co-Immunoprecipitation Assay

    Linker histone H1.2 attenuates the ATM-dependent DNA damage response. a Immunoblots for H1.2, H1.3 and H1.4 protein levels in wild-type, H1.2, H1.3 or H1.4 KO HeLa cells. 1# and 2# indicate two clones which were generated using different sgRNAs. b Wild-type, H1.2, H1.3 or H1.4 KO (1#) HeLa cells were treated with 40 μM etoposide for 0, 30 and 60 min and analyzed by immunoblotting. c Wild-type and H1.2 KO (1#) HeLa cells were transfected with the indicated plasmids with or without exposure to 10 Gy IR and analyzed by immunoblotting 1 h post IR. d HeLa cells were transfected with GFP-H1.2 and exposed to 10 Gy irradiation (IR) with or without 2 h prior exposure to 2 μM Ku57788. Cells were collected 1 h post IR and subjected to immunofluorescent assay. Cells with > 5 γ-H2AX foci were counted. The data represent the mean ± SD. Scale bars, 10 μm. e HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. f A-T cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 1 h and analyzed by immunoblotting. g , h Wild type and H1.2 KO (1#) HeLa cells were mixed and then treated with 40 μM etoposide for 2 h or left untreated (Ctr) and analyzed by immunofluorescence. The intensity of ATM or phospho-ATM S1981 in the etoposide-treated wild-type cells was normalized to 1. The arrows indicate representative cells. All data represent the mean ± SD. Scale bars, 10 μm
    Figure Legend Snippet: Linker histone H1.2 attenuates the ATM-dependent DNA damage response. a Immunoblots for H1.2, H1.3 and H1.4 protein levels in wild-type, H1.2, H1.3 or H1.4 KO HeLa cells. 1# and 2# indicate two clones which were generated using different sgRNAs. b Wild-type, H1.2, H1.3 or H1.4 KO (1#) HeLa cells were treated with 40 μM etoposide for 0, 30 and 60 min and analyzed by immunoblotting. c Wild-type and H1.2 KO (1#) HeLa cells were transfected with the indicated plasmids with or without exposure to 10 Gy IR and analyzed by immunoblotting 1 h post IR. d HeLa cells were transfected with GFP-H1.2 and exposed to 10 Gy irradiation (IR) with or without 2 h prior exposure to 2 μM Ku57788. Cells were collected 1 h post IR and subjected to immunofluorescent assay. Cells with > 5 γ-H2AX foci were counted. The data represent the mean ± SD. Scale bars, 10 μm. e HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. f A-T cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 1 h and analyzed by immunoblotting. g , h Wild type and H1.2 KO (1#) HeLa cells were mixed and then treated with 40 μM etoposide for 2 h or left untreated (Ctr) and analyzed by immunofluorescence. The intensity of ATM or phospho-ATM S1981 in the etoposide-treated wild-type cells was normalized to 1. The arrows indicate representative cells. All data represent the mean ± SD. Scale bars, 10 μm

    Techniques Used: Western Blot, Clone Assay, Generated, Transfection, Irradiation, Immunofluorescence

    Linker histone H1.2 interacts with ATM and directly inhibits its activity. a An N-terminal GST-p53 (1–99 aa) peptide was used as a substrate for in vitro phosphorylation assay with or without HIS-H1.2/H1.4. b HCT116 cells were transfected with FLAG-H1.2 or an empty vector and mononucleosomes were extracted and subjected to in vitro phosphorylation. c GST alone or GST-ATM fragments were incubated with HIS-H1.2 for the GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-ATM fragment 7 (F7, 1239–1770 aa) for GST pull-down assay. * indicates specific protein bands. e Free histones extracted from HeLa cells were used as substrates for in vitro phosphorylation in the presence of GST-H1.2 fragments or GST alone. The relative intensity of γ-H2AX/H2AX was calculated. * indicates specific protein bands. f Total HeLa cell lysates were immunoprecipitated with anti-ATM or anti-IgG antibodies. The precipitated proteins were analyzed by mass spectrometry after SDS-PAGE electrophoresis and silver staining. Name in bold indicates the desired protein. g HEK293T cells were transfected with the indicated plasmids and subjected to Co-IP assay with FLAG-conjugated M2 beads. h HeLa cells were transfected with FLAG-H1.2 or an empty vector and treated as indicated with 40 μM etoposide for 2 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. i ATM was immunoprecipitated using FLAG-conjugated M2 beads in HEK293T cells overexpressed with FLAG-ATM and incubated with (−) or without (+) recombinant HIS-H1.2 in kinase buffer. Recombinant ATM substrates, including HIS-H2AX (full-length) and GST-p53 (amino acids 1–99) were incubated without ATP. The interacting proteins were eluted and analyzed by immunoblotting
    Figure Legend Snippet: Linker histone H1.2 interacts with ATM and directly inhibits its activity. a An N-terminal GST-p53 (1–99 aa) peptide was used as a substrate for in vitro phosphorylation assay with or without HIS-H1.2/H1.4. b HCT116 cells were transfected with FLAG-H1.2 or an empty vector and mononucleosomes were extracted and subjected to in vitro phosphorylation. c GST alone or GST-ATM fragments were incubated with HIS-H1.2 for the GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-ATM fragment 7 (F7, 1239–1770 aa) for GST pull-down assay. * indicates specific protein bands. e Free histones extracted from HeLa cells were used as substrates for in vitro phosphorylation in the presence of GST-H1.2 fragments or GST alone. The relative intensity of γ-H2AX/H2AX was calculated. * indicates specific protein bands. f Total HeLa cell lysates were immunoprecipitated with anti-ATM or anti-IgG antibodies. The precipitated proteins were analyzed by mass spectrometry after SDS-PAGE electrophoresis and silver staining. Name in bold indicates the desired protein. g HEK293T cells were transfected with the indicated plasmids and subjected to Co-IP assay with FLAG-conjugated M2 beads. h HeLa cells were transfected with FLAG-H1.2 or an empty vector and treated as indicated with 40 μM etoposide for 2 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. i ATM was immunoprecipitated using FLAG-conjugated M2 beads in HEK293T cells overexpressed with FLAG-ATM and incubated with (−) or without (+) recombinant HIS-H1.2 in kinase buffer. Recombinant ATM substrates, including HIS-H2AX (full-length) and GST-p53 (amino acids 1–99) were incubated without ATP. The interacting proteins were eluted and analyzed by immunoblotting

    Techniques Used: Activity Assay, In Vitro, Phosphorylation Assay, Transfection, Plasmid Preparation, Incubation, Pull Down Assay, Immunoprecipitation, Mass Spectrometry, SDS Page, Electrophoresis, Silver Staining, Co-Immunoprecipitation Assay, Recombinant

    Linker histone H1.2 is rapidly displaced from chromatin and degraded upon DNA damage. a , b HeLa cells were exposed to 10 Gy irradiation (IR) and released at the indicated time or treated with 20 μM etoposide for the indicated time. Chromatin was fractionated and subjected to immunoblotting. c DR-GFP U2OS cells were transfected with I- Sce I endonuclease or an empty vector and subjected to chromatin immunoprecipitation with the indicated antibodies followed by real-time PCR 48 h post transfection. All data represent the mean ± SD. d HeLa cells were transfected with GFP-H1.2 and subjected to laser micro-irradiation-coupled live-cell imaging. The initial signal intensity of GFP-H1.2 was normalized to 1 and ~15 IR paths from 10 separate cells were calculated. All data represent the mean ± SD. Scale bars, 10 μm. e HeLa cells were treated with either 1 μM adriamycin at 1 μM, 20 μM etoposide, 10 μM cisplatin, 2 mM hydroxyurea or 10 μM oxaliplatin for 12 h and analyzed by immunoblotting. f HeLa cells were treated with 1 μM MG132 or 50 μM CHQ for 12 h with or without 20 μM etoposide and analyzed by immunoblotting. g HeLa cells were treated with etoposide at 20 μM for 12 h with or without Oprozomib at 100 nM and analyzed by immunoblotting. h HeLa cells were treated with 1 μM TSA for the indicated time or to increasing concentrations of sodium butyrate (NaB) for 12 h and analyzed by immunoblotting. A pan-ac H3 antibodies was used as a positive control. i HeLa cells were exposed to increasing concentrations of sodium chloride (NaCl) for 2 h and analyzed by immunoblotting
    Figure Legend Snippet: Linker histone H1.2 is rapidly displaced from chromatin and degraded upon DNA damage. a , b HeLa cells were exposed to 10 Gy irradiation (IR) and released at the indicated time or treated with 20 μM etoposide for the indicated time. Chromatin was fractionated and subjected to immunoblotting. c DR-GFP U2OS cells were transfected with I- Sce I endonuclease or an empty vector and subjected to chromatin immunoprecipitation with the indicated antibodies followed by real-time PCR 48 h post transfection. All data represent the mean ± SD. d HeLa cells were transfected with GFP-H1.2 and subjected to laser micro-irradiation-coupled live-cell imaging. The initial signal intensity of GFP-H1.2 was normalized to 1 and ~15 IR paths from 10 separate cells were calculated. All data represent the mean ± SD. Scale bars, 10 μm. e HeLa cells were treated with either 1 μM adriamycin at 1 μM, 20 μM etoposide, 10 μM cisplatin, 2 mM hydroxyurea or 10 μM oxaliplatin for 12 h and analyzed by immunoblotting. f HeLa cells were treated with 1 μM MG132 or 50 μM CHQ for 12 h with or without 20 μM etoposide and analyzed by immunoblotting. g HeLa cells were treated with etoposide at 20 μM for 12 h with or without Oprozomib at 100 nM and analyzed by immunoblotting. h HeLa cells were treated with 1 μM TSA for the indicated time or to increasing concentrations of sodium butyrate (NaB) for 12 h and analyzed by immunoblotting. A pan-ac H3 antibodies was used as a positive control. i HeLa cells were exposed to increasing concentrations of sodium chloride (NaCl) for 2 h and analyzed by immunoblotting

    Techniques Used: Irradiation, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Live Cell Imaging, Positive Control

    Linker histone H1.2 inhibits ATM recruitment and activation by interacting with MRN. a Wild type and H1.2 KO (1#) HeLa cells were transfected with GFP-NBS1 and subjected to laser micro-irradiation-coupled live-cell imaging. Images were taken every 10 s for 10 min and the relative intensity of the irradiation path signal was shown. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells extracts were analyzed by Co-IP assay with or without benzonase treatment with the indicated antibodies. c GST alone or GST-MRE11, RAD50 and NBS1 were incubated with HIS-H1.2 for GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-MRE11 for GST pull-down assay. * indicates specific protein bands. e Wild type or NBS1 KO HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. f HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. g , h HeLa cells were transfected with the indicated plasmids, and the whole cell lysates were immunoprecipitated with ATM antibody and analyzed by immunoblotting. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 2 h. Whole cell extracts were prepared and analyzed by Co-IP assay and immunoblotting with the indicated antibodies
    Figure Legend Snippet: Linker histone H1.2 inhibits ATM recruitment and activation by interacting with MRN. a Wild type and H1.2 KO (1#) HeLa cells were transfected with GFP-NBS1 and subjected to laser micro-irradiation-coupled live-cell imaging. Images were taken every 10 s for 10 min and the relative intensity of the irradiation path signal was shown. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells extracts were analyzed by Co-IP assay with or without benzonase treatment with the indicated antibodies. c GST alone or GST-MRE11, RAD50 and NBS1 were incubated with HIS-H1.2 for GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-MRE11 for GST pull-down assay. * indicates specific protein bands. e Wild type or NBS1 KO HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. f HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 2 h and analyzed by immunoblotting. g , h HeLa cells were transfected with the indicated plasmids, and the whole cell lysates were immunoprecipitated with ATM antibody and analyzed by immunoblotting. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 2 h. Whole cell extracts were prepared and analyzed by Co-IP assay and immunoblotting with the indicated antibodies

    Techniques Used: Activation Assay, Transfection, Irradiation, Live Cell Imaging, Co-Immunoprecipitation Assay, Incubation, Pull Down Assay, Immunoprecipitation

    14) Product Images from "Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway"

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    Journal: Biochemical Journal

    doi: 10.1042/BJ20031705

    The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).
    Figure Legend Snippet: The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).

    Techniques Used: Activation Assay, Incubation, Activity Assay

    Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).
    Figure Legend Snippet: Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay

    The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.
    Figure Legend Snippet: The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.

    Techniques Used: Expressing, Incubation, Activation Assay, SDS Page

    The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).
    Figure Legend Snippet: The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).

    Techniques Used: Incubation, Activity Assay

    Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).
    Figure Legend Snippet: Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).

    Techniques Used: Incubation, Activity Assay

    Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).
    Figure Legend Snippet: Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).

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

    Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).
    Figure Legend Snippet: Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).

    Techniques Used: Activation Assay, Activity Assay

    15) Product Images from "The putative oncotarget CSN5 controls a transcription-uncorrelated p53-mediated autophagy implicated in cancer cell survival under curcumin treatment"

    Article Title: The putative oncotarget CSN5 controls a transcription-uncorrelated p53-mediated autophagy implicated in cancer cell survival under curcumin treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11940

    Interruption of CSN5/p53-induced autophagy pathway does not sensitize normal cells to curcumin-induced apoptosis ( A ) Representative Western blot images in BJ cells treated with curcumin for 2, 4, 6 and 8 h. ( B ) Representative images of autophagy detection in BJ cells treated with curcumin for 2, 4, 6 and 8 h. ( C ) Representative images of autophagy detection in BJ cells pre-transfected with CSN5 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( D ) Representative Western blot images in BJ cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( E ) Representative images of autophagy detection were showed in BJ cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( F ) Representative Western blot images in BJ cells transfected with CSN5 siRNA or control siRNA, or combination of CSN5 and p53 siRNA for 48 h. ( G ) Representative images of autophagy detection in (F) were showed. ( H ) Representative Western blot images in BJ cells pre-transfected with p53 or ATG5 siRNA or control siRNA for 48 h, then treated with curcumin for 6 h or etoposide for 12 h. ( I ) Cell number was determined by CCK-8 assay in BJ cells transfected with control, p53 or ATG5 siRNA for 48 h, and then treated with curcumin for 6 h. Scale bar: 10 μm.
    Figure Legend Snippet: Interruption of CSN5/p53-induced autophagy pathway does not sensitize normal cells to curcumin-induced apoptosis ( A ) Representative Western blot images in BJ cells treated with curcumin for 2, 4, 6 and 8 h. ( B ) Representative images of autophagy detection in BJ cells treated with curcumin for 2, 4, 6 and 8 h. ( C ) Representative images of autophagy detection in BJ cells pre-transfected with CSN5 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( D ) Representative Western blot images in BJ cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( E ) Representative images of autophagy detection were showed in BJ cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( F ) Representative Western blot images in BJ cells transfected with CSN5 siRNA or control siRNA, or combination of CSN5 and p53 siRNA for 48 h. ( G ) Representative images of autophagy detection in (F) were showed. ( H ) Representative Western blot images in BJ cells pre-transfected with p53 or ATG5 siRNA or control siRNA for 48 h, then treated with curcumin for 6 h or etoposide for 12 h. ( I ) Cell number was determined by CCK-8 assay in BJ cells transfected with control, p53 or ATG5 siRNA for 48 h, and then treated with curcumin for 6 h. Scale bar: 10 μm.

    Techniques Used: Western Blot, Transfection, CCK-8 Assay

    Curcumin controls p53 to induce autophagy uncorrelated to its transcriptional activity ( A ) Transcriptional activity analysis of p53 in HCT116 wt , HCT116 p53−/− and HT29 cancer cells. ( B ) Representative Western blot images in HT29 cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( C ) Representative Western blot images in HCT116 wt , HCT116 p53−/− and HT29 cells treated with curcumin for 6 h. ( D ) Representative images of autophagy detection were showed in HCT116 wt , HCT116 p53−/− and HT29 cells treated with curcumin for 6 h. ( E ) Transcriptional activity analysis of p53 in HCT116 p53−/− cancer cells infected with lentivirus expressing flag-p53 or flag-p53 R273H tag fusion. ( F ) Representative Western blot images in HCT116 p53−/− cells pre-infected with lentivirus expressing flag-p53 and flag-p53 R273H tag fusion, and then treated with curcumin for 6 h. ( G ) Representative images of autophagy detection in (F) were showed. ( H ) Transcriptional activity of p53 in HepG2 cells pre-treated with PFTα for 6 h and then treated with curcumin for 6 h or etoposide for 12 h. ( I ) Representative Western blot images in HepG2 cells pre-treated with PFTα for 6 h, and then treated with curcumin for 6 h. ( J ) Representative images of autophagy detection in (I) were showed. Scale bar: 10 μm.
    Figure Legend Snippet: Curcumin controls p53 to induce autophagy uncorrelated to its transcriptional activity ( A ) Transcriptional activity analysis of p53 in HCT116 wt , HCT116 p53−/− and HT29 cancer cells. ( B ) Representative Western blot images in HT29 cells pre-transfected with p53 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( C ) Representative Western blot images in HCT116 wt , HCT116 p53−/− and HT29 cells treated with curcumin for 6 h. ( D ) Representative images of autophagy detection were showed in HCT116 wt , HCT116 p53−/− and HT29 cells treated with curcumin for 6 h. ( E ) Transcriptional activity analysis of p53 in HCT116 p53−/− cancer cells infected with lentivirus expressing flag-p53 or flag-p53 R273H tag fusion. ( F ) Representative Western blot images in HCT116 p53−/− cells pre-infected with lentivirus expressing flag-p53 and flag-p53 R273H tag fusion, and then treated with curcumin for 6 h. ( G ) Representative images of autophagy detection in (F) were showed. ( H ) Transcriptional activity of p53 in HepG2 cells pre-treated with PFTα for 6 h and then treated with curcumin for 6 h or etoposide for 12 h. ( I ) Representative Western blot images in HepG2 cells pre-treated with PFTα for 6 h, and then treated with curcumin for 6 h. ( J ) Representative images of autophagy detection in (I) were showed. Scale bar: 10 μm.

    Techniques Used: Activity Assay, Western Blot, Transfection, Infection, Expressing

    The effect of curcumin on CSN5 and p53 ( A ) Representative Western blot images in HepG2 cells treated with curcumin for 6 h, etoposide for 12 h, 5-FU for 12 h or cisplatin for 12 h. ( B ) Representative Western blot images in HepG2 and BJ cells pre-transfected with CSN5 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( C ) Representative Western blot images in HepG2 cells pre-infected with lentivirus expressing JAB1-V5 tag fusion, and then treated with curcumin for 6 h. ( D ) Representative Western blot images in HepG2 cells treated with curcumin or etoposide for 3, 6, 9, 12, 15 or 18 h, respectively. ( E ) Representative Western blot images in HepG2 cells pre-treated with caffeine for 6 h, and then treated with curcumin for 6 h or etoposide for 12 h, respectively. ( F ) p53 transcriptional activity was detected by luciferase reporter assay in HepG2 cells treated with curcumin or etoposide for 3, 6, 9, 12, 15 or 18 h. ( G ) RT-PCR analysis of p21 expression levels in HepG2 cells treated with curcumin or etoposide for 12 h. Data were the mean value of 3 independent experiments. Values are expressed as the mean ± SEM, n = 3, * p
    Figure Legend Snippet: The effect of curcumin on CSN5 and p53 ( A ) Representative Western blot images in HepG2 cells treated with curcumin for 6 h, etoposide for 12 h, 5-FU for 12 h or cisplatin for 12 h. ( B ) Representative Western blot images in HepG2 and BJ cells pre-transfected with CSN5 siRNA or control siRNA for 48 h, and then treated with curcumin for 6 h. ( C ) Representative Western blot images in HepG2 cells pre-infected with lentivirus expressing JAB1-V5 tag fusion, and then treated with curcumin for 6 h. ( D ) Representative Western blot images in HepG2 cells treated with curcumin or etoposide for 3, 6, 9, 12, 15 or 18 h, respectively. ( E ) Representative Western blot images in HepG2 cells pre-treated with caffeine for 6 h, and then treated with curcumin for 6 h or etoposide for 12 h, respectively. ( F ) p53 transcriptional activity was detected by luciferase reporter assay in HepG2 cells treated with curcumin or etoposide for 3, 6, 9, 12, 15 or 18 h. ( G ) RT-PCR analysis of p21 expression levels in HepG2 cells treated with curcumin or etoposide for 12 h. Data were the mean value of 3 independent experiments. Values are expressed as the mean ± SEM, n = 3, * p

    Techniques Used: Western Blot, Transfection, Infection, Expressing, Activity Assay, Luciferase, Reporter Assay, Reverse Transcription Polymerase Chain Reaction

    16) Product Images from "Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes"

    Article Title: Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s120506520

    Calibration curve families. Cyclic voltammetric measurements, in PBS, obtained for all drugs in their pharmacological range and etoposide at fixed concentrations (0–25–50–75–100 μM). Cytochrome P4503A4 was used for ifosfamide ( A ), cytochrome P4503A4 for cyclophosphamide ( B ), and cytochrome P4501A2 for ftorafur ( C ). Calibration curves for etoposide detection in its pharmacological range obtained with a CNT-electrode in presence of ifosfamide at fixed concentrations (0–40–80–120–160 μM) are illustrated in ( D ).
    Figure Legend Snippet: Calibration curve families. Cyclic voltammetric measurements, in PBS, obtained for all drugs in their pharmacological range and etoposide at fixed concentrations (0–25–50–75–100 μM). Cytochrome P4503A4 was used for ifosfamide ( A ), cytochrome P4503A4 for cyclophosphamide ( B ), and cytochrome P4501A2 for ftorafur ( C ). Calibration curves for etoposide detection in its pharmacological range obtained with a CNT-electrode in presence of ifosfamide at fixed concentrations (0–40–80–120–160 μM) are illustrated in ( D ).

    Techniques Used:

    Cyclic voltammetric responses (on the left) and calibration curves (on the right), obtained from cyclic voltammetric measurements in PBS with increasing aliquots of drugs: CNT-electrode for etoposide ( A ), cytochrome P4503A4 for ifosfamide ( B ), cytochrome P4502B6 for cyclophosphamide ( C ), and P4501A2 for ftorafur ( D ). For the CNT-electrode the zoom in the oxidation peak at 220 mV is shown ( A , on the left). For the electrodes modified with cytochromes we report the zoom of the reduction peak at −450 mV ( B, C , and D , on the left). The measured drugs concentration falls in their pharmacological range. The voltammograms were acquired with drugs dissolved in PBS and at the scan rate of 20 mV/s.
    Figure Legend Snippet: Cyclic voltammetric responses (on the left) and calibration curves (on the right), obtained from cyclic voltammetric measurements in PBS with increasing aliquots of drugs: CNT-electrode for etoposide ( A ), cytochrome P4503A4 for ifosfamide ( B ), cytochrome P4502B6 for cyclophosphamide ( C ), and P4501A2 for ftorafur ( D ). For the CNT-electrode the zoom in the oxidation peak at 220 mV is shown ( A , on the left). For the electrodes modified with cytochromes we report the zoom of the reduction peak at −450 mV ( B, C , and D , on the left). The measured drugs concentration falls in their pharmacological range. The voltammograms were acquired with drugs dissolved in PBS and at the scan rate of 20 mV/s.

    Techniques Used: Modification, Concentration Assay

    Comparison between cyclic voltammograms obtained with bare electrode (black curve) and CNT-nanostructured electrode (grey curve), in presence of etoposide 100 μM. The peak at around −200 mV is due to the oxygen moieties derived from carbon-nanotubes as reported in [ 5 , 47 ]. Two oxidation peaks at +220 mV and +450 mV and two reduction peaks at +150 mV and +350 mV are visible. These data confirm the peaks reported in literature [ 48 ], obtained through etoposide cyclic voltammetry at glassy carbon electrode.
    Figure Legend Snippet: Comparison between cyclic voltammograms obtained with bare electrode (black curve) and CNT-nanostructured electrode (grey curve), in presence of etoposide 100 μM. The peak at around −200 mV is due to the oxygen moieties derived from carbon-nanotubes as reported in [ 5 , 47 ]. Two oxidation peaks at +220 mV and +450 mV and two reduction peaks at +150 mV and +350 mV are visible. These data confirm the peaks reported in literature [ 48 ], obtained through etoposide cyclic voltammetry at glassy carbon electrode.

    Techniques Used: Derivative Assay

    17) Product Images from "Hsp27 as a Negative Regulator of Cytochrome c Release"

    Article Title: Hsp27 as a Negative Regulator of Cytochrome c Release

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.3.816-834.2002

    Scheme of Hsp27-induced effects on apoptosis. Hsp27 reduces F-actin damage induced by apoptotic drugs (e.g., cytochalasin D and staurosporine) and thus attenuates the activation of the pathway that links F-actin damages to mitochondria. Activation of this pathway induces cytochrome c release, apoptosome formation, and procaspase activation. The mechanism of activation of this pathway is unknown but may be a consequence of altered integrin signaling pathway or changes in F-actin-dependent subcellular distribution of members of the Bcl-2 family such as Bid. Hsp27 also attenuates cytochrome c release in cells exposed to agents that do not rapidly destroy F-actin architecture (e.g., etoposide and Fas), suggesting that other upstream pathways are under the control of Hsp27 expression. Hsp27 also acts downstream of mitochondria by interfering with apoptosome formation, probably through its binding to cytochrome c once it is released from mitochondria. Hsp27 also appears to bind and negatively modulate caspase 3. In L929 cells, the upstream activity necessitates a higher level of Hsp27 expression ( > 0.45 ng/μg) compared to the downstream effect which is already detected in cells expressing Hsp27 (0.1 ng/μg).
    Figure Legend Snippet: Scheme of Hsp27-induced effects on apoptosis. Hsp27 reduces F-actin damage induced by apoptotic drugs (e.g., cytochalasin D and staurosporine) and thus attenuates the activation of the pathway that links F-actin damages to mitochondria. Activation of this pathway induces cytochrome c release, apoptosome formation, and procaspase activation. The mechanism of activation of this pathway is unknown but may be a consequence of altered integrin signaling pathway or changes in F-actin-dependent subcellular distribution of members of the Bcl-2 family such as Bid. Hsp27 also attenuates cytochrome c release in cells exposed to agents that do not rapidly destroy F-actin architecture (e.g., etoposide and Fas), suggesting that other upstream pathways are under the control of Hsp27 expression. Hsp27 also acts downstream of mitochondria by interfering with apoptosome formation, probably through its binding to cytochrome c once it is released from mitochondria. Hsp27 also appears to bind and negatively modulate caspase 3. In L929 cells, the upstream activity necessitates a higher level of Hsp27 expression ( > 0.45 ng/μg) compared to the downstream effect which is already detected in cells expressing Hsp27 (0.1 ng/μg).

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

    Hsp27 expression interferes with the staurosporine- and etoposide-induced Bid intracellular relocalization in murine L929 cells. Control L929-C2 and Hsp27-expressing L929-Hsp27 cells kept either untreated (NT) or exposed to staurosporine (1 μM) or etoposide (500 μM) for either 1, 2, or 4 h were lysed under conditions which preserve mitochondrial and membrane integrity as described in Materials and Methods. The resulting P20 and S cytosolic fractions were analyzed in immunoblots probed with anti-Bid antibody. Autoradiographs of immunoblots are shown.
    Figure Legend Snippet: Hsp27 expression interferes with the staurosporine- and etoposide-induced Bid intracellular relocalization in murine L929 cells. Control L929-C2 and Hsp27-expressing L929-Hsp27 cells kept either untreated (NT) or exposed to staurosporine (1 μM) or etoposide (500 μM) for either 1, 2, or 4 h were lysed under conditions which preserve mitochondrial and membrane integrity as described in Materials and Methods. The resulting P20 and S cytosolic fractions were analyzed in immunoblots probed with anti-Bid antibody. Autoradiographs of immunoblots are shown.

    Techniques Used: Expressing, Western Blot

    Phalloidin counteracts the cytochalasin D-mediated release of cytochrome c from mitochondria and procaspase 3 activation. The effect is partial in case of staurosporine-treated cells. (A) Cytochrome c release analysis. Control (L929-C2) cells were either kept untreated or treated for 6 h with 0.5 μM cytochalasin D in the absence (−) or presence (+) of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Cells were then processed for cytochrome c release analysis and proteins present in the different fractions were analyzed in immunoblots as described in Materials and Methods. The presence of cytochrome c (Cytc) and Hsc70 in the different fractions is shown. Autoradiographs of ECL-revealed immunoblots are presented. Note that phalloidin strongly decreases the release of cytochrome c induced by cytochalasin D. Lanes, P, pellet from untreated cells; lanes 0 and 6, soluble fractions isolated from untreated cells (0) or cells treated for 6 h with cytochalasin D. (B) Same as panel A, but in this case cells were treated with 1 μM staurosporine. (C) Caspase 3 activation in L929-C2 extracts isolated after 6 h of treatment with 0.5 μM cytochalasin D in the absence or presence of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Activity of DEVD-specific caspases was then measured using the fluorescent substrate DEVD-AFC as described in Materials and Methods. (D) Same as panel C but in the presence of 1 μM staurosporine. (E) Same as panel C but in the presence of 500 μM etoposide. The activation index was determined as the ratio between the activity in extracts of treated cells to that measured in extracts of nontreated cells. The histogram shown is representative of three identical experiments; standard deviations (error bars) are presented ( n = 3). Note the protective activity of phalloidin.
    Figure Legend Snippet: Phalloidin counteracts the cytochalasin D-mediated release of cytochrome c from mitochondria and procaspase 3 activation. The effect is partial in case of staurosporine-treated cells. (A) Cytochrome c release analysis. Control (L929-C2) cells were either kept untreated or treated for 6 h with 0.5 μM cytochalasin D in the absence (−) or presence (+) of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Cells were then processed for cytochrome c release analysis and proteins present in the different fractions were analyzed in immunoblots as described in Materials and Methods. The presence of cytochrome c (Cytc) and Hsc70 in the different fractions is shown. Autoradiographs of ECL-revealed immunoblots are presented. Note that phalloidin strongly decreases the release of cytochrome c induced by cytochalasin D. Lanes, P, pellet from untreated cells; lanes 0 and 6, soluble fractions isolated from untreated cells (0) or cells treated for 6 h with cytochalasin D. (B) Same as panel A, but in this case cells were treated with 1 μM staurosporine. (C) Caspase 3 activation in L929-C2 extracts isolated after 6 h of treatment with 0.5 μM cytochalasin D in the absence or presence of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Activity of DEVD-specific caspases was then measured using the fluorescent substrate DEVD-AFC as described in Materials and Methods. (D) Same as panel C but in the presence of 1 μM staurosporine. (E) Same as panel C but in the presence of 500 μM etoposide. The activation index was determined as the ratio between the activity in extracts of treated cells to that measured in extracts of nontreated cells. The histogram shown is representative of three identical experiments; standard deviations (error bars) are presented ( n = 3). Note the protective activity of phalloidin.

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

    Hsp27 expression interferes with cytochalasin D- and staurosporine-induced damages to F-actin. Fluorescence photomicrographs demonstrating the effect of cytochalasin D and staurosporine on F-actin fibers. Control L929-C2 cells (A to D) and human Hsp27-expressing L929-Hsp27 cells (E to H) were plated on glass plates and allowed to enter exponential cell growth phase for 24 h. They were then either kept untreated (A and E) or treated with 0.5 μM cytochalasin D (B and F), 1 μM staurosporine (C and G), or 500 μM etoposide (D and H). After 2 h of treatment the cells were fixed, stained with FITC-labeled phalloidin, and examined under a fluorescence microscope. The photomicrograph in panel G is enlarged in order to better detect the F-actin fibers (arrows), which are still visible in L929-Hsp27 cells treated with staurosporine. Bar, 10 μM.
    Figure Legend Snippet: Hsp27 expression interferes with cytochalasin D- and staurosporine-induced damages to F-actin. Fluorescence photomicrographs demonstrating the effect of cytochalasin D and staurosporine on F-actin fibers. Control L929-C2 cells (A to D) and human Hsp27-expressing L929-Hsp27 cells (E to H) were plated on glass plates and allowed to enter exponential cell growth phase for 24 h. They were then either kept untreated (A and E) or treated with 0.5 μM cytochalasin D (B and F), 1 μM staurosporine (C and G), or 500 μM etoposide (D and H). After 2 h of treatment the cells were fixed, stained with FITC-labeled phalloidin, and examined under a fluorescence microscope. The photomicrograph in panel G is enlarged in order to better detect the F-actin fibers (arrows), which are still visible in L929-Hsp27 cells treated with staurosporine. Bar, 10 μM.

    Techniques Used: Expressing, Fluorescence, Staining, Labeling, Microscopy

    18) Product Images from "hMOF Acetylation of DBC1/CCAR2 Prevents Binding and Inhibition of SirT1"

    Article Title: hMOF Acetylation of DBC1/CCAR2 Prevents Binding and Inhibition of SirT1

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00874-13

    DNA damage inhibits DBC1-hMOF binding and DBC1 acetylation. (a) U2OS cells were treated with 50 μM etoposide (Eto) for 6 h and subjected to IP and Western blot analysis to detect the binding between endogenous hMOF-DBC1 (top panels) and SirT1-DBC1
    Figure Legend Snippet: DNA damage inhibits DBC1-hMOF binding and DBC1 acetylation. (a) U2OS cells were treated with 50 μM etoposide (Eto) for 6 h and subjected to IP and Western blot analysis to detect the binding between endogenous hMOF-DBC1 (top panels) and SirT1-DBC1

    Techniques Used: Binding Assay, Western Blot

    19) Product Images from "Prostate cancer radiosensitization through PARP-1 hyperactivation"

    Article Title: Prostate cancer radiosensitization through PARP-1 hyperactivation

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-1418

    β-Lap-induced, NQO1-mediated ROS formation and SSBs are required for cell death in human prostate cancer cells. A , Relative survival of PC-3 cells after β-lap treatment in the presence or absence of dicoumarol (Dic, 40 μMol/L). Data are means ± SE for three independent experiments performed in sextuplicate. B , Reactive Oxygen Species (ROS) formation was indirectly monitored using the Oxidized Glutathione (GSSG) recycling assay in β-lap-exposed PC-3 cells in the presence or absence of 40 μMol/L Dic. Graphed results were means ± SE and represented three experiments performed in duplicate. C, Left panel, alkaline vs. neutral comet assays to assess total DNA damage or DNA double strand breaks (DSBs), respectively. H 2 O 2 and Etoposide were used as positive controls for agents causing mostly SSBs or DSBs, respectively (not shown). Right panel, DNA damage assessment (arbitrary units (AU) of comet tail lengths) using NIH Image J software. Top graph as indicated, bottom graph (alkaline conditions), Data are means ± SE from 100 cells. D , DNA damage in PC-3 cells after IR (20 Gy) versus β-lap (4 μMol/L, 2h; left panel). Comet tail lengths (arbitrary units, AU) assessed using NIH Image J software. Data are means ± SE from 100 cells (right panel). Statistics (t-tests) were performed *** p
    Figure Legend Snippet: β-Lap-induced, NQO1-mediated ROS formation and SSBs are required for cell death in human prostate cancer cells. A , Relative survival of PC-3 cells after β-lap treatment in the presence or absence of dicoumarol (Dic, 40 μMol/L). Data are means ± SE for three independent experiments performed in sextuplicate. B , Reactive Oxygen Species (ROS) formation was indirectly monitored using the Oxidized Glutathione (GSSG) recycling assay in β-lap-exposed PC-3 cells in the presence or absence of 40 μMol/L Dic. Graphed results were means ± SE and represented three experiments performed in duplicate. C, Left panel, alkaline vs. neutral comet assays to assess total DNA damage or DNA double strand breaks (DSBs), respectively. H 2 O 2 and Etoposide were used as positive controls for agents causing mostly SSBs or DSBs, respectively (not shown). Right panel, DNA damage assessment (arbitrary units (AU) of comet tail lengths) using NIH Image J software. Top graph as indicated, bottom graph (alkaline conditions), Data are means ± SE from 100 cells. D , DNA damage in PC-3 cells after IR (20 Gy) versus β-lap (4 μMol/L, 2h; left panel). Comet tail lengths (arbitrary units, AU) assessed using NIH Image J software. Data are means ± SE from 100 cells (right panel). Statistics (t-tests) were performed *** p

    Techniques Used: Software

    20) Product Images from "In vitro evaluation of the antitumor effect of bismuth lipophilic nanoparticles (BisBAL NPs) on breast cancer cells"

    Article Title: In vitro evaluation of the antitumor effect of bismuth lipophilic nanoparticles (BisBAL NPs) on breast cancer cells

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S179095

    Detection of apoptosis in human breast cancer cells by BisBAL NPs. Notes: Apoptosis induction in MCF-7 cells by BisBAL NPs was detected with the CF ® 488A Annexin V and 7-AAD apoptosis assay. Etoposide at 100 µM was used as a positive control of an apoptotic agent, whereas pure culture medium was used as a negative control; 1 and 10 µM BisBAL NPs were analyzed on MCF-7 cells. After treatments, cell cultures were observed with a fluorescence microscope using fluorescein isothiocyanate (green) and CY5 (red) filters. Bar, 5 µm.
    Figure Legend Snippet: Detection of apoptosis in human breast cancer cells by BisBAL NPs. Notes: Apoptosis induction in MCF-7 cells by BisBAL NPs was detected with the CF ® 488A Annexin V and 7-AAD apoptosis assay. Etoposide at 100 µM was used as a positive control of an apoptotic agent, whereas pure culture medium was used as a negative control; 1 and 10 µM BisBAL NPs were analyzed on MCF-7 cells. After treatments, cell cultures were observed with a fluorescence microscope using fluorescein isothiocyanate (green) and CY5 (red) filters. Bar, 5 µm.

    Techniques Used: Apoptosis Assay, Positive Control, Negative Control, Fluorescence, Microscopy

    Genotoxic effect of BisBAL NPs on MCF-7 breast cancer cells. Notes: MCF-7 cells were incubated for 24 hours with culture medium as a growth control of intact cells, 100 µM etoposide (positive control of genotoxic effect), and 1 or 10 µM BisBAL NPs. DNA damage was evaluated with the OxiSelect ™ Comet Assay Kit, and DAPI-stained DNA was observed with an epifluorescence microscope with DAPI filter. Red arrow indicates the stellar morphology. Bar, 5 µm. Abbreviation: BisBAL NPs, lipophilic bismuth nanoparticles.
    Figure Legend Snippet: Genotoxic effect of BisBAL NPs on MCF-7 breast cancer cells. Notes: MCF-7 cells were incubated for 24 hours with culture medium as a growth control of intact cells, 100 µM etoposide (positive control of genotoxic effect), and 1 or 10 µM BisBAL NPs. DNA damage was evaluated with the OxiSelect ™ Comet Assay Kit, and DAPI-stained DNA was observed with an epifluorescence microscope with DAPI filter. Red arrow indicates the stellar morphology. Bar, 5 µm. Abbreviation: BisBAL NPs, lipophilic bismuth nanoparticles.

    Techniques Used: Incubation, Positive Control, Single Cell Gel Electrophoresis, Staining, Microscopy

    21) Product Images from "Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer"

    Article Title: Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0030420

    Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.
    Figure Legend Snippet: Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean ± SD for each concentration.

    Techniques Used: Activity Assay, MTT Assay, Concentration Assay

    22) Product Images from "Surgical delivery of drug releasing poly(lactic-co-glycolic acid)/poly(ethylene glycol) paste with in vivo effects against glioblastoma"

    Article Title: Surgical delivery of drug releasing poly(lactic-co-glycolic acid)/poly(ethylene glycol) paste with in vivo effects against glioblastoma

    Journal: Annals of The Royal College of Surgeons of England

    doi: 10.1308/003588414X13946184903568

    Animal weights after surgical implantation of blank poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG), PLGA/PEG with 80mg/kg etoposide and PLGA/PEG with 160mg/kg etoposide
    Figure Legend Snippet: Animal weights after surgical implantation of blank poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG), PLGA/PEG with 80mg/kg etoposide and PLGA/PEG with 160mg/kg etoposide

    Techniques Used:

    Immunohistochemistry in murine flank xenografts A D: Staining for Ki-67 proliferation marker in a U87 murine flank xenograft partially resected and treated with blank poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG) (A) or PLGA/PEG loaded with 160mg/kg etoposide (D), showing reduction in tumour cell proliferation in response to active etoposide release B E: Staining for vascular endothelial growth factor (VEGF) in a U87 murine flank xenograft partially resected and treated with blank PLGA/PEG (B) or PLGA/PEG loaded with 160mg/kg etoposide (E), showing reduction in VEGF positivity C F: Staining for the angiogenic vessel marker endoglin, showing new vessel formation in a U87 murine flank xenograft treated with blank PLGA/PEG (C) but not for PLGA/PEG loaded with 160mg/kg etoposide (F) G: Low power view of an excised U87 murine flank xenograft treated with PLGA/PEG loaded with 160mg/kg etoposide showing the inner zone of necrosis, more viable tumour centrally and the outer capsule of normal mouse tissue H: Immunostaining for endoglin in a U87 murine flank xenograft treated with PLGA/PEG loaded with 160mg/kg etoposide showing damaged blood vessels in highly necrotic tissue I: Immunostaining for VEGF with tumour cells growing around blank PLGA/PEG particles showing the non-toxic nature of the carrier biomaterial
    Figure Legend Snippet: Immunohistochemistry in murine flank xenografts A D: Staining for Ki-67 proliferation marker in a U87 murine flank xenograft partially resected and treated with blank poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG) (A) or PLGA/PEG loaded with 160mg/kg etoposide (D), showing reduction in tumour cell proliferation in response to active etoposide release B E: Staining for vascular endothelial growth factor (VEGF) in a U87 murine flank xenograft partially resected and treated with blank PLGA/PEG (B) or PLGA/PEG loaded with 160mg/kg etoposide (E), showing reduction in VEGF positivity C F: Staining for the angiogenic vessel marker endoglin, showing new vessel formation in a U87 murine flank xenograft treated with blank PLGA/PEG (C) but not for PLGA/PEG loaded with 160mg/kg etoposide (F) G: Low power view of an excised U87 murine flank xenograft treated with PLGA/PEG loaded with 160mg/kg etoposide showing the inner zone of necrosis, more viable tumour centrally and the outer capsule of normal mouse tissue H: Immunostaining for endoglin in a U87 murine flank xenograft treated with PLGA/PEG loaded with 160mg/kg etoposide showing damaged blood vessels in highly necrotic tissue I: Immunostaining for VEGF with tumour cells growing around blank PLGA/PEG particles showing the non-toxic nature of the carrier biomaterial

    Techniques Used: Immunohistochemistry, Staining, Marker, Immunostaining

    In vivo effects of drug eluting poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG) A: Tumour growth following partial tumour resection and implantation of blank PLGA/PEG, PLGA/PEG with 80mg/kg etoposide and PLGA/PEG with 160mg/kg etoposide. The higher dose PLGA/PEG with etoposide demonstrated reduction in tumour growth in the animals until day 21, when in vitro studies predicted drug release would stop. By the day 28 measurement, tumour growth had resumed at a rapid rate. B: Sample bioluminescent image of two mice with partial resection of a U373 flank xenograft, mouse on left with blank PLGA/PEG and mouse on right with PLGA/PEG loaded with 320mg/kg etoposide, showing considerable reduction in bioluminescence and corresponding reduction in tumour load
    Figure Legend Snippet: In vivo effects of drug eluting poly(lactic- co -glycolic acid)/poly(ethylene glycol) (PLGA/PEG) A: Tumour growth following partial tumour resection and implantation of blank PLGA/PEG, PLGA/PEG with 80mg/kg etoposide and PLGA/PEG with 160mg/kg etoposide. The higher dose PLGA/PEG with etoposide demonstrated reduction in tumour growth in the animals until day 21, when in vitro studies predicted drug release would stop. By the day 28 measurement, tumour growth had resumed at a rapid rate. B: Sample bioluminescent image of two mice with partial resection of a U373 flank xenograft, mouse on left with blank PLGA/PEG and mouse on right with PLGA/PEG loaded with 320mg/kg etoposide, showing considerable reduction in bioluminescence and corresponding reduction in tumour load

    Techniques Used: In Vivo, In Vitro, Mouse Assay

    23) Product Images from "Unmodified Histone H3K4 and DNA-Dependent Protein Kinase Recruit Autoimmune Regulator to Target Genes"

    Article Title: Unmodified Histone H3K4 and DNA-Dependent Protein Kinase Recruit Autoimmune Regulator to Target Genes

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06359-11

    Kinase activity of DNA-PKcs is not required for effects of AIRE in cells. (a) Catalytic activity of DNA-PKcs is dispensable for the AIRE-induced expression of the KRT14 gene. KRT14 mRNA levels were analyzed in 293T cells, which transiently expressed AIRE or the empty plasmid vector in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). (b) Inhibition of DNA-PKcs prevents its autophosphorylation after the induction of double-strand DNA breaks. After the standard incubation period, etoposide (ETO) was added for 1 h to 293T cells in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). Levels of DNA-PKcs autophosphorylated on serine at position 2056 (S2056P) were quantified relative to those of total DNA-PKcs and are presented in numbers below the panels.
    Figure Legend Snippet: Kinase activity of DNA-PKcs is not required for effects of AIRE in cells. (a) Catalytic activity of DNA-PKcs is dispensable for the AIRE-induced expression of the KRT14 gene. KRT14 mRNA levels were analyzed in 293T cells, which transiently expressed AIRE or the empty plasmid vector in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). (b) Inhibition of DNA-PKcs prevents its autophosphorylation after the induction of double-strand DNA breaks. After the standard incubation period, etoposide (ETO) was added for 1 h to 293T cells in the presence or absence of 1 μM Nu7441 (iDNA-PKcs). Levels of DNA-PKcs autophosphorylated on serine at position 2056 (S2056P) were quantified relative to those of total DNA-PKcs and are presented in numbers below the panels.

    Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Inhibition, Incubation

    24) Product Images from "YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress"

    Article Title: YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress

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

    doi: 10.1073/pnas.0402283101

    YY1 inhibits the activation of p53 in response to genotoxic stress. ( A ) ChIP analysis of the p21 promoter after DNA damage. U2OS cells were infected with control (GFP) or YY1 adenovirus and either left untreated or treated with etoposide (ETOP, 5 μM)
    Figure Legend Snippet: YY1 inhibits the activation of p53 in response to genotoxic stress. ( A ) ChIP analysis of the p21 promoter after DNA damage. U2OS cells were infected with control (GFP) or YY1 adenovirus and either left untreated or treated with etoposide (ETOP, 5 μM)

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Infection

    Inactivation of YY1 promotes apoptosis in response to DNA damage. ( A ) siRNA-mediated inactivation of YY1. U2OS cells were transfected with control (GL2) or YY1 siRNA (20 nM) and treated with etoposide (5 μM) for the indicated times. The expression
    Figure Legend Snippet: Inactivation of YY1 promotes apoptosis in response to DNA damage. ( A ) siRNA-mediated inactivation of YY1. U2OS cells were transfected with control (GL2) or YY1 siRNA (20 nM) and treated with etoposide (5 μM) for the indicated times. The expression

    Techniques Used: Transfection, Expressing

    25) Product Images from "Neuroprotective Role of the Reaper-Related Serine Protease HtrA2/Omi Revealed by Targeted Deletion in Mice"

    Article Title: Neuroprotective Role of the Reaper-Related Serine Protease HtrA2/Omi Revealed by Targeted Deletion in Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9848-9862.2004

    Deletion of HtrA2/Omi results in increased sensitivity to mitochondrion-damaging agents. Thymocytes were isolated from control and HtrA2/Omi knockout animals and incubated with increasing concentrations of anti-Fas antibody (A), etoposide (B), or the mitochondrion-damaging agents CCCP (C) and rotenone (D). Viability was determined 16 h after treatment by flow cytometry using propidium iodide. Results are representative of three independent experiments. (E) Viability of simian virus 40 large-T-antigen-immortalized MEFs derived from wild-type (+/+) and HtrA2/Omi knockout animals, determined by measurement of sub-G 1 cell populations by flow cytometry. Cells were incubated in the presence of CCCP (25 μM for 27 h), rotenone (25 μM for 27 h), tunicamycin (2.5 μg/ml for 27 h), or hydrogen peroxide (3 μM for 4.5 h) and compared to untreated control cells. Results show the means ± standard deviations of results of three independent experiments. (F) The neuronalresponse to glutamate-induced cytotoxicity was determined in primary neurons isolated from E14.5 embryos cultured in vitro for 10 days. Following incubation with the indicated concentrations of glutamate for 4 h, cells were stained with Hoechst 33342 and nuclear morphology was determined. Results show the means ± standard deviations of results from two independent experiments where six fields with at least 40 cells were scored for each data point.
    Figure Legend Snippet: Deletion of HtrA2/Omi results in increased sensitivity to mitochondrion-damaging agents. Thymocytes were isolated from control and HtrA2/Omi knockout animals and incubated with increasing concentrations of anti-Fas antibody (A), etoposide (B), or the mitochondrion-damaging agents CCCP (C) and rotenone (D). Viability was determined 16 h after treatment by flow cytometry using propidium iodide. Results are representative of three independent experiments. (E) Viability of simian virus 40 large-T-antigen-immortalized MEFs derived from wild-type (+/+) and HtrA2/Omi knockout animals, determined by measurement of sub-G 1 cell populations by flow cytometry. Cells were incubated in the presence of CCCP (25 μM for 27 h), rotenone (25 μM for 27 h), tunicamycin (2.5 μg/ml for 27 h), or hydrogen peroxide (3 μM for 4.5 h) and compared to untreated control cells. Results show the means ± standard deviations of results of three independent experiments. (F) The neuronalresponse to glutamate-induced cytotoxicity was determined in primary neurons isolated from E14.5 embryos cultured in vitro for 10 days. Following incubation with the indicated concentrations of glutamate for 4 h, cells were stained with Hoechst 33342 and nuclear morphology was determined. Results show the means ± standard deviations of results from two independent experiments where six fields with at least 40 cells were scored for each data point.

    Techniques Used: Isolation, Knock-Out, Incubation, Flow Cytometry, Cytometry, Derivative Assay, Cell Culture, In Vitro, Staining

    Analysis of HtrA2/Omi Smac/DIABLO double-knockout animals. (A) Detection of the wild-type and mutant HtrA2/Omi and Smac/DIABLO loci in the offspring of animals with a heterozygous deletion of the genes for both HtrA2/Omi and Smac/DIABLO. PCR was performed on genomic tail DNA templates. (B) Average body weights of Smac/DIABLO +/+ HtrA2/Omi +/+ (wild-type [wt]) and Smac/DIABLO −/− HtrA2/Omi −/− (double-knockout [KO]) mice. (C and D) The sensitivities of wild-type and double-knockout MEFs to TNF-α (C), and etoposide (D) are shown. Viability was determined 16 h following treatment by flow cytometry using propidium iodide. Results show the means ± standard deviations of results from three independent experiments.
    Figure Legend Snippet: Analysis of HtrA2/Omi Smac/DIABLO double-knockout animals. (A) Detection of the wild-type and mutant HtrA2/Omi and Smac/DIABLO loci in the offspring of animals with a heterozygous deletion of the genes for both HtrA2/Omi and Smac/DIABLO. PCR was performed on genomic tail DNA templates. (B) Average body weights of Smac/DIABLO +/+ HtrA2/Omi +/+ (wild-type [wt]) and Smac/DIABLO −/− HtrA2/Omi −/− (double-knockout [KO]) mice. (C and D) The sensitivities of wild-type and double-knockout MEFs to TNF-α (C), and etoposide (D) are shown. Viability was determined 16 h following treatment by flow cytometry using propidium iodide. Results show the means ± standard deviations of results from three independent experiments.

    Techniques Used: Double Knockout, Mutagenesis, Polymerase Chain Reaction, Mouse Assay, Flow Cytometry, Cytometry

    26) Product Images from "Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells"

    Article Title: Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells

    Journal: Oncotarget

    doi:

    γH2AX phosphorylation labeling, cell cycle and cell viability analysis 72 h after treatment (a) The percentages of positive cells for γH2AX phosphorylation are reported for siRNA at 100 nM (si Luc , white bars, si DNMT1 (6) hatched bars and si DNMT1 (7) stripped bars) alone or in the presence of 100 nM DAC; for DAC alone at 10, 30 and 100 nM (gray bars) or for etoposide at 0.5 and 5 μM (black bars). P -value: ** ≤ 0.01 and *** ≤ 0.001. (b) The cell cycle repartition, indicated as percentages of cells in each phase (G0/G1 lower bars, S middle bars, G2/M upper bars), is shown for the various treatments applied to the cells. The stars on the histogram refer to the p -values calculated for both G0/G1 and G2/M compartments in KG1 cells treated with 100 nM DAC combined with either DNMT1 siRNA1(6) and 1(7) versus the cells exposed to 100 nM DAC combined with the control siRNA ( Luc ). (c) After γH2AX phosphorylation measurement, cells from the same batch were seeded for another 4 days and cell viability assessed. The percentage of viable cells is indicated relative to their respective controls (si Luc and 0 nM DAC) set to 100% viable cells. White bars: 100 nM si Luc ; hatched bars: 100 nM si DNMT1 (6); stripped bars: 100 nM DNMT1 (7); gray bars: DAC at 10, 30 and 100 nM.
    Figure Legend Snippet: γH2AX phosphorylation labeling, cell cycle and cell viability analysis 72 h after treatment (a) The percentages of positive cells for γH2AX phosphorylation are reported for siRNA at 100 nM (si Luc , white bars, si DNMT1 (6) hatched bars and si DNMT1 (7) stripped bars) alone or in the presence of 100 nM DAC; for DAC alone at 10, 30 and 100 nM (gray bars) or for etoposide at 0.5 and 5 μM (black bars). P -value: ** ≤ 0.01 and *** ≤ 0.001. (b) The cell cycle repartition, indicated as percentages of cells in each phase (G0/G1 lower bars, S middle bars, G2/M upper bars), is shown for the various treatments applied to the cells. The stars on the histogram refer to the p -values calculated for both G0/G1 and G2/M compartments in KG1 cells treated with 100 nM DAC combined with either DNMT1 siRNA1(6) and 1(7) versus the cells exposed to 100 nM DAC combined with the control siRNA ( Luc ). (c) After γH2AX phosphorylation measurement, cells from the same batch were seeded for another 4 days and cell viability assessed. The percentage of viable cells is indicated relative to their respective controls (si Luc and 0 nM DAC) set to 100% viable cells. White bars: 100 nM si Luc ; hatched bars: 100 nM si DNMT1 (6); stripped bars: 100 nM DNMT1 (7); gray bars: DAC at 10, 30 and 100 nM.

    Techniques Used: Labeling

    Effect of combining DNMT1 siRNA and DAC on global DNA methylation and on the methylation of LINE-1 and AluSc repeated sequences (a) DNMT1 Western blot performed on KG1 cells exposed to combination of siRNA ( Luc , DNMT1 (6) or DNMT1 (7)) and two doses of DAC (10 and 100 nM) (left), compared to DAC-treated cells at 10, 30 or 100 nM (right). The full gel images are accessible in Supplementary Figure S6 . The percentages of DNMT1 protein level (% DNMT1) normalized to 100% for their respective control (si Luc for electroporated cells and non treated for DAC treatment) are indicated. The image shown is representative of two independent experiments, except for the siRNA 1(7) that was tested in duplicates in this experiment (b) 72 h post-treatment, cells were collected and total DNA methylation was measured by flow cytometry for cells treated with siRNA Luc (100 nM) alone or combined with 10 or 100 nM DAC (white bars), siRNA DNMT1 (6) (100 nM) alone or combined with 10 or 100 nM DAC (hatched bars); siRNA DNMT1 (7) (100 nM) alone or combined with 10 or 100 nM DAC (striped bars); DAC alone at 10, 30 and 100 nM (gray bars) or etoposide at 0.5 or 5 μM (black bars). P value: * ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001. (c–d) KG1 cells treated for 72 hours with various combinations of DNMT1 siRNA (1(6) and 1(7)) and DAC were analyzed by bisulfite conversion followed by pyrosequencing for methylation changes in LINE-1 (c) and AluSc (d). Cells were treated also with increasing concentration of etoposide. The mean methylation level of all CpG present in the sequenced DNA and the standard error are represented. The percentage of demethylation (normalized to 100% for the controls, i.e . luciferase siRNA and untreated cells) are also indicated. FM, fully methylated DNA control, and UM, unmethylated DNA control.
    Figure Legend Snippet: Effect of combining DNMT1 siRNA and DAC on global DNA methylation and on the methylation of LINE-1 and AluSc repeated sequences (a) DNMT1 Western blot performed on KG1 cells exposed to combination of siRNA ( Luc , DNMT1 (6) or DNMT1 (7)) and two doses of DAC (10 and 100 nM) (left), compared to DAC-treated cells at 10, 30 or 100 nM (right). The full gel images are accessible in Supplementary Figure S6 . The percentages of DNMT1 protein level (% DNMT1) normalized to 100% for their respective control (si Luc for electroporated cells and non treated for DAC treatment) are indicated. The image shown is representative of two independent experiments, except for the siRNA 1(7) that was tested in duplicates in this experiment (b) 72 h post-treatment, cells were collected and total DNA methylation was measured by flow cytometry for cells treated with siRNA Luc (100 nM) alone or combined with 10 or 100 nM DAC (white bars), siRNA DNMT1 (6) (100 nM) alone or combined with 10 or 100 nM DAC (hatched bars); siRNA DNMT1 (7) (100 nM) alone or combined with 10 or 100 nM DAC (striped bars); DAC alone at 10, 30 and 100 nM (gray bars) or etoposide at 0.5 or 5 μM (black bars). P value: * ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001. (c–d) KG1 cells treated for 72 hours with various combinations of DNMT1 siRNA (1(6) and 1(7)) and DAC were analyzed by bisulfite conversion followed by pyrosequencing for methylation changes in LINE-1 (c) and AluSc (d). Cells were treated also with increasing concentration of etoposide. The mean methylation level of all CpG present in the sequenced DNA and the standard error are represented. The percentage of demethylation (normalized to 100% for the controls, i.e . luciferase siRNA and untreated cells) are also indicated. FM, fully methylated DNA control, and UM, unmethylated DNA control.

    Techniques Used: DNA Methylation Assay, Methylation, Western Blot, Flow Cytometry, Cytometry, Concentration Assay, Luciferase

    Impact of combining DNMT1 siRNA with DAC on promoter methylation of CDH1 and CDKN2B, and promoter methylation and expression of TP73 CDH1 (a) , CDKN2B (b) and TP73 (c) methylation was measured by MS-HRM 72 h after treating the cells with DNMT siRNA Luc (100 nM, white bars), siRNA DNMT1 (6) at 100 nM (hatched bars), siRNA DNMT1 (7) at 100 nM (stripped bars) and in combination with DAC (10 and 100 nM) (left), compared to DAC alone at 10, 30 and 100 nM (gray bars) or etoposide at 0.5 and 5 μM (black bars) (right). The mean percentage values of methylation, relative to the respective controls are indicated. (d) qRT-PCR analysis of TP73 mRNA expression of the above treated cells. The expression ratio of the controls was normalized to 1, i.e ., luciferase siRNA (for electroporated cells), water (the solvent of DAC) and DMSO (the solvent of etoposide), respectively. P value: * ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001.
    Figure Legend Snippet: Impact of combining DNMT1 siRNA with DAC on promoter methylation of CDH1 and CDKN2B, and promoter methylation and expression of TP73 CDH1 (a) , CDKN2B (b) and TP73 (c) methylation was measured by MS-HRM 72 h after treating the cells with DNMT siRNA Luc (100 nM, white bars), siRNA DNMT1 (6) at 100 nM (hatched bars), siRNA DNMT1 (7) at 100 nM (stripped bars) and in combination with DAC (10 and 100 nM) (left), compared to DAC alone at 10, 30 and 100 nM (gray bars) or etoposide at 0.5 and 5 μM (black bars) (right). The mean percentage values of methylation, relative to the respective controls are indicated. (d) qRT-PCR analysis of TP73 mRNA expression of the above treated cells. The expression ratio of the controls was normalized to 1, i.e ., luciferase siRNA (for electroporated cells), water (the solvent of DAC) and DMSO (the solvent of etoposide), respectively. P value: * ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001.

    Techniques Used: Methylation, Expressing, Mass Spectrometry, Quantitative RT-PCR, Luciferase

    27) Product Images from "UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells"

    Article Title: UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12372

    UBXN2A and CHIP proteins form a ternary complex with mot‐2. (A) Schematic drawings of the protein structures of mot‐2 and the CHIP E3 ubiquitin ligase. Mot‐2 has two major domains, an N‐terminal ATPase domain (ATP) and a C‐terminal substrate‐binding domain (SBD). These two domains are reciprocally controlled by the presence of ATP/ADP on the ATP domain and a client protein bound to the SBD (Dores‐Silva et al ). (B) HeLa cell lysates were subjected to IP using anti‐mot‐2 antibodies. IP experiments showed mot‐2 can pull down CHIP. (C) WB analysis was used to verify the lack of the CHIP protein in CHIP knockout mice using small intestine (SI) and large intestine (LI) tissue lysates. To verify the mot‐2‐CHIP complex in vivo , colon tissue (LI) lysates from C57Bl/6 WT (CHIP +/+ ) or CHIP knockout (CHIP −/− ) were subjected to IP using anti‐CHIP antibodies immobilized on magnetic IgA. WB showed mot‐2 protein can be pulled down only from WT colon lysates where CHIP proteins are present (HC, heavy chain of immunoglobulins). (D) HCT‐116 cells were exposed to stress using the genotoxic stress agent etoposide for 24 h followed by IP experiments with anti‐UBXN2A antibodies immobilized on magnetic beads coupled with protein A. Control groups received DMSO treatment. WB analysis showed interaction of CHIP and UBXN2A takes place in the cytoplasm and not the nucleus due to the absence of CHIP in the nucleus. More importantly, we observed genotoxic stress induces nucleo‐cytoplasmic translocation of UBXN2A, which leads to an increase in UBXN2A binding to CHIP in cytoplasmic fraction. (E) In another set of experiments, DMSO‐ and etoposide (Eto)‐treated cells were subjected to IP using anti‐CHIP antibodies. WB results show the existence of a triple complex containing mot‐2, CHIP, and UBXN2A. More importantly, E indicates etoposide enhances the association of CHIP and mot‐2 protein alongside an elevation of UBXN2A in the cytoplasm, as previously described. Collectively, the experiments conducted in this figure indicate UBXN2A, mot‐2, and CHIP proteins can be present simultaneously in a multiprotein complex.
    Figure Legend Snippet: UBXN2A and CHIP proteins form a ternary complex with mot‐2. (A) Schematic drawings of the protein structures of mot‐2 and the CHIP E3 ubiquitin ligase. Mot‐2 has two major domains, an N‐terminal ATPase domain (ATP) and a C‐terminal substrate‐binding domain (SBD). These two domains are reciprocally controlled by the presence of ATP/ADP on the ATP domain and a client protein bound to the SBD (Dores‐Silva et al ). (B) HeLa cell lysates were subjected to IP using anti‐mot‐2 antibodies. IP experiments showed mot‐2 can pull down CHIP. (C) WB analysis was used to verify the lack of the CHIP protein in CHIP knockout mice using small intestine (SI) and large intestine (LI) tissue lysates. To verify the mot‐2‐CHIP complex in vivo , colon tissue (LI) lysates from C57Bl/6 WT (CHIP +/+ ) or CHIP knockout (CHIP −/− ) were subjected to IP using anti‐CHIP antibodies immobilized on magnetic IgA. WB showed mot‐2 protein can be pulled down only from WT colon lysates where CHIP proteins are present (HC, heavy chain of immunoglobulins). (D) HCT‐116 cells were exposed to stress using the genotoxic stress agent etoposide for 24 h followed by IP experiments with anti‐UBXN2A antibodies immobilized on magnetic beads coupled with protein A. Control groups received DMSO treatment. WB analysis showed interaction of CHIP and UBXN2A takes place in the cytoplasm and not the nucleus due to the absence of CHIP in the nucleus. More importantly, we observed genotoxic stress induces nucleo‐cytoplasmic translocation of UBXN2A, which leads to an increase in UBXN2A binding to CHIP in cytoplasmic fraction. (E) In another set of experiments, DMSO‐ and etoposide (Eto)‐treated cells were subjected to IP using anti‐CHIP antibodies. WB results show the existence of a triple complex containing mot‐2, CHIP, and UBXN2A. More importantly, E indicates etoposide enhances the association of CHIP and mot‐2 protein alongside an elevation of UBXN2A in the cytoplasm, as previously described. Collectively, the experiments conducted in this figure indicate UBXN2A, mot‐2, and CHIP proteins can be present simultaneously in a multiprotein complex.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Western Blot, Knock-Out, Mouse Assay, In Vivo, Magnetic Beads, Translocation Assay

    28) Product Images from "Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network"

    Article Title: Kinesin light chain 4 as a new target for lung cancer chemoresistance via targeted inhibition of checkpoint kinases in the DNA repair network

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2592-z

    KLC4 was related to chemoresistance in vitro. a Cell viability was measured every 12 h after treatment with 10 μM cisplatin. b Death of cells treated with or without 10 μM cisplatin during 12 h intervals. c After cisplatin treatment for 24 h, the cells were fixed with 4% paraformaldehyde and immunostained using an antibody targeting γH2AX (DNA damage marker); DNA was visualized using DAPI staining. d Protein levels of KLC4, cleaved PARP, and active caspase-3 were determined using western blotting after cisplatin treatment. e – g Cells treated with etoposide, gefitinib, or Taxol, respectively. Twenty-four hours after the drug treatments, the cells were analyzed using cell viability assay, followed by ELISA.
    Figure Legend Snippet: KLC4 was related to chemoresistance in vitro. a Cell viability was measured every 12 h after treatment with 10 μM cisplatin. b Death of cells treated with or without 10 μM cisplatin during 12 h intervals. c After cisplatin treatment for 24 h, the cells were fixed with 4% paraformaldehyde and immunostained using an antibody targeting γH2AX (DNA damage marker); DNA was visualized using DAPI staining. d Protein levels of KLC4, cleaved PARP, and active caspase-3 were determined using western blotting after cisplatin treatment. e – g Cells treated with etoposide, gefitinib, or Taxol, respectively. Twenty-four hours after the drug treatments, the cells were analyzed using cell viability assay, followed by ELISA.

    Techniques Used: In Vitro, Marker, Staining, Western Blot, Viability Assay, Enzyme-linked Immunosorbent Assay

    KLC4 depletion reversed chemoresistance in lung cancer cells. a Viability of R-H460 cells treated with or without 10 μM cisplatin after transfection with siCON (negative control) or siKLC4. b Cell death in R-H460 cells [treated as described in ( a )] using annexin V/propidium iodide staining. c Protein levels of KLC4, cleaved PARP, and active caspase-3 (cell death marker) as determined using western blotting. d Viability of R-H460 cells treated with or without 10 μM etoposide after transfection with siCON or siKLC4. e − f R-H460 cells were treated with or without 10 µM etoposide after transfection with KLC4 siRNA. Cell death was measured 48 h after treatment using annexin V/propidium iodide staining ( e ), and western blotting ( f ).
    Figure Legend Snippet: KLC4 depletion reversed chemoresistance in lung cancer cells. a Viability of R-H460 cells treated with or without 10 μM cisplatin after transfection with siCON (negative control) or siKLC4. b Cell death in R-H460 cells [treated as described in ( a )] using annexin V/propidium iodide staining. c Protein levels of KLC4, cleaved PARP, and active caspase-3 (cell death marker) as determined using western blotting. d Viability of R-H460 cells treated with or without 10 μM etoposide after transfection with siCON or siKLC4. e − f R-H460 cells were treated with or without 10 µM etoposide after transfection with KLC4 siRNA. Cell death was measured 48 h after treatment using annexin V/propidium iodide staining ( e ), and western blotting ( f ).

    Techniques Used: Transfection, Negative Control, Staining, Marker, Western Blot

    KLC4 knockdown induced activation of CHK1/CHK2 in R-H460 cells. a Twenty-four hours after transfection with siCON (negative control) or siKLC4, the cells were fixed with paraformaldehyde and immunostained using antibodies targeting p-CHK1 and p-CHK2. b Cell lysates [from cells treated as in (a)] were prepared and used for immunoblotting with antibodies against p-CHK1 (S345), CHK1, p-CHK2 (T68), and CHK2. c Protein levels of KLC4, p-p53 (S15), p53, and Noxa, as determined using western blotting after transfection with KLC4 siRNA. d , e Cells were treated with or without 10 μM cisplatin ( d ) or 10 μM etoposide ( e ) after transfection with KLC4 siRNA. The cell lysates were used for immunoblotting with antibodies against KLC4, p-CHK1 (S345), CHK1, p-CHK2 (T68), and CHK2.
    Figure Legend Snippet: KLC4 knockdown induced activation of CHK1/CHK2 in R-H460 cells. a Twenty-four hours after transfection with siCON (negative control) or siKLC4, the cells were fixed with paraformaldehyde and immunostained using antibodies targeting p-CHK1 and p-CHK2. b Cell lysates [from cells treated as in (a)] were prepared and used for immunoblotting with antibodies against p-CHK1 (S345), CHK1, p-CHK2 (T68), and CHK2. c Protein levels of KLC4, p-p53 (S15), p53, and Noxa, as determined using western blotting after transfection with KLC4 siRNA. d , e Cells were treated with or without 10 μM cisplatin ( d ) or 10 μM etoposide ( e ) after transfection with KLC4 siRNA. The cell lysates were used for immunoblotting with antibodies against KLC4, p-CHK1 (S345), CHK1, p-CHK2 (T68), and CHK2.

    Techniques Used: Activation Assay, Transfection, Negative Control, Western Blot

    Downregulation of KLC4 induced DNA damage response. a Cells were fixed with 4% paraformaldehyde and stained using antibodies targeting KLC4 and γH2AX; DNA was visualized using DAPI staining. b Protein levels of KLC4 and γH2AX were determined using western blotting after transfection with KLC4 siRNA. c R-H460 cells were treated with 10 µM cisplatin after transfection with KLC4 siRNA for 24 h. Cells were fixed and immunostained using antibodies targeting KLC4 and γH2AX. d , e R-H460 cells were pre-treated with 10 µM cisplatin (d) or 10 µM etoposide ( e ) and transfected with siKLC4. The cell lysates were prepared and used for immunoblotting with antibodies against KLC4 and γH2AX. f , g Reporter cells were transfected with KLC4 siRNA and pCB-ASce vector (HA tagged I-SceI expression). GFP-positive cells were counted using flow cytometry in the U2OS EJ-GFP cell ( f ) or the U2OS DR-GFP cell ( g ).
    Figure Legend Snippet: Downregulation of KLC4 induced DNA damage response. a Cells were fixed with 4% paraformaldehyde and stained using antibodies targeting KLC4 and γH2AX; DNA was visualized using DAPI staining. b Protein levels of KLC4 and γH2AX were determined using western blotting after transfection with KLC4 siRNA. c R-H460 cells were treated with 10 µM cisplatin after transfection with KLC4 siRNA for 24 h. Cells were fixed and immunostained using antibodies targeting KLC4 and γH2AX. d , e R-H460 cells were pre-treated with 10 µM cisplatin (d) or 10 µM etoposide ( e ) and transfected with siKLC4. The cell lysates were prepared and used for immunoblotting with antibodies against KLC4 and γH2AX. f , g Reporter cells were transfected with KLC4 siRNA and pCB-ASce vector (HA tagged I-SceI expression). GFP-positive cells were counted using flow cytometry in the U2OS EJ-GFP cell ( f ) or the U2OS DR-GFP cell ( g ).

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

    29) Product Images from "The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus"

    Article Title: The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq011

    Plk5 expression is induced following different stress stimuli. ( A ) Murine NIH 3T3 cells were either left untreated (control) or treated for 18 h with the DNA damaging agents etoposide (Etop) or HU, the spindle disassembly agent, nocodazole (Noc) or were serum starved (SS). ( B ) Human HEK293 cells were either left untreated (control) or treated with DNA damaging agent etoposide or doxorubicin for 6 or 24 h. The Plk5 mRNA level was measured by qPCR in both cases.
    Figure Legend Snippet: Plk5 expression is induced following different stress stimuli. ( A ) Murine NIH 3T3 cells were either left untreated (control) or treated for 18 h with the DNA damaging agents etoposide (Etop) or HU, the spindle disassembly agent, nocodazole (Noc) or were serum starved (SS). ( B ) Human HEK293 cells were either left untreated (control) or treated with DNA damaging agent etoposide or doxorubicin for 6 or 24 h. The Plk5 mRNA level was measured by qPCR in both cases.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Human and mouse Plk5 proteins are both expressed in cultured cells. Western blot showing that mouse Plk5 ( A ) and human Plk5 ( B ) proteins are detected in lysates from NIH3T3 and HEK-293 cells, respectively, using a Plk5 polyclonal antibody. ( C ) Immunofluorescence detection of endogenous mPlk5 in discrete foci (arrows) in NIH3T3 cells. ( D ) A shRNA that targets hPlk5 mRNA was able to deplete Plk5 protein from HEK-293 cells. ( E ) HEK-293 cells transiently transfected with vector expressing both shRNA against Plk5 and GFP were analyzed by flow cytometry. The cell cycle distribution of GFP-positive cells is shown in left top corner of each panel. Depletion of hPlk5 from HEK-293cells leads to loss of G2/M checkpoint in response to treatment with the DNA damaging agent, etoposide (lower panel, right) compared to nondepleted cells (lower panel, left).
    Figure Legend Snippet: Human and mouse Plk5 proteins are both expressed in cultured cells. Western blot showing that mouse Plk5 ( A ) and human Plk5 ( B ) proteins are detected in lysates from NIH3T3 and HEK-293 cells, respectively, using a Plk5 polyclonal antibody. ( C ) Immunofluorescence detection of endogenous mPlk5 in discrete foci (arrows) in NIH3T3 cells. ( D ) A shRNA that targets hPlk5 mRNA was able to deplete Plk5 protein from HEK-293 cells. ( E ) HEK-293 cells transiently transfected with vector expressing both shRNA against Plk5 and GFP were analyzed by flow cytometry. The cell cycle distribution of GFP-positive cells is shown in left top corner of each panel. Depletion of hPlk5 from HEK-293cells leads to loss of G2/M checkpoint in response to treatment with the DNA damaging agent, etoposide (lower panel, right) compared to nondepleted cells (lower panel, left).

    Techniques Used: Cell Culture, Western Blot, Immunofluorescence, shRNA, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    Plk5 transcriptional activation in response to DNA damage is not p53-dependent. ( A ) p53 consensus binding sites in Plk5 promoter, ( B ) mPlk5 mRNA expression levels measured by qPCR following etoposide and pifithrin treatment of NIH 3T3 cells for 18 h. ( C ) Expression of p21 protein in lysates from cells either nontreated ( 1 ), treated with pifithrin ( 2 ), etoposide alone ( 3 ) or pifithrin and etoposide ( 4 ) was analyzed by western blot.
    Figure Legend Snippet: Plk5 transcriptional activation in response to DNA damage is not p53-dependent. ( A ) p53 consensus binding sites in Plk5 promoter, ( B ) mPlk5 mRNA expression levels measured by qPCR following etoposide and pifithrin treatment of NIH 3T3 cells for 18 h. ( C ) Expression of p21 protein in lysates from cells either nontreated ( 1 ), treated with pifithrin ( 2 ), etoposide alone ( 3 ) or pifithrin and etoposide ( 4 ) was analyzed by western blot.

    Techniques Used: Activation Assay, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    30) Product Images from "Chk2 prevents mitotic exit when the majority of kinetochores are unattached"

    Article Title: Chk2 prevents mitotic exit when the majority of kinetochores are unattached

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201310071

    Chk2 localizes to kinetochores in unperturbed early prometaphase. (A) Localization of Chk2. (B) Localization of phosphorylated Chk2-T383 (pT383). Boxed values show mean pT383/Hec1 fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. (A and B) Bars, 5 µm. The insets show 1.7× magnification of kinetochores. (C and D) Western blot analysis of total pT383, Chk2 phospho-T68 (pT68), Chk2, and actin. Cells were untreated (un) or treated with taxol, 3.32 µM nocodazole (nocod), or etoposide for 8 h followed by shake-off as indicated. Values at untreated were taken as 1. MI, mitotic index; NS, nonspecific. (E) Chk2 in vitro kinase assay. inh II, inhibitor II. (top) Autoradiography analysis of Aurora B substrates. (bottom) Ponceau staining.
    Figure Legend Snippet: Chk2 localizes to kinetochores in unperturbed early prometaphase. (A) Localization of Chk2. (B) Localization of phosphorylated Chk2-T383 (pT383). Boxed values show mean pT383/Hec1 fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. (A and B) Bars, 5 µm. The insets show 1.7× magnification of kinetochores. (C and D) Western blot analysis of total pT383, Chk2 phospho-T68 (pT68), Chk2, and actin. Cells were untreated (un) or treated with taxol, 3.32 µM nocodazole (nocod), or etoposide for 8 h followed by shake-off as indicated. Values at untreated were taken as 1. MI, mitotic index; NS, nonspecific. (E) Chk2 in vitro kinase assay. inh II, inhibitor II. (top) Autoradiography analysis of Aurora B substrates. (bottom) Ponceau staining.

    Techniques Used: Fluorescence, Western Blot, In Vitro, Kinase Assay, Autoradiography, Staining

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

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

    Journal: Molecular Biology of the Cell

    doi:

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

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

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

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

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

    Techniques Used: Labeling

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

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

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

    Techniques Used:

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

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

    32) Product Images from "MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts"

    Article Title: MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201504005

    MRN and CtIP remove Top2 adducts during S phase . (A) Mock or CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated in the presence of 100 µM etoposide for 40 min. Reactions were stopped and diluted in denaturing buffer, and genomic DNA was fractionated via CsCl gradients. The fractions were transferred and probed with anti- Xenopus Top2 antibodies. (B) Relative quantification of three independent experiments as shown in A. Error bars indicate standard deviations (*, P = 0.021, two-tailed unpaired t test). (C) CtIP, Mre11, or CtIP/Mre11 immunodepletion from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (D) Quantification of Top2–DNA adducts in Mre11-depleted, CtIP-depleted, or MRE11- and CtIP-depleted extracts as described in A. (*, P
    Figure Legend Snippet: MRN and CtIP remove Top2 adducts during S phase . (A) Mock or CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated in the presence of 100 µM etoposide for 40 min. Reactions were stopped and diluted in denaturing buffer, and genomic DNA was fractionated via CsCl gradients. The fractions were transferred and probed with anti- Xenopus Top2 antibodies. (B) Relative quantification of three independent experiments as shown in A. Error bars indicate standard deviations (*, P = 0.021, two-tailed unpaired t test). (C) CtIP, Mre11, or CtIP/Mre11 immunodepletion from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (D) Quantification of Top2–DNA adducts in Mre11-depleted, CtIP-depleted, or MRE11- and CtIP-depleted extracts as described in A. (*, P

    Techniques Used: Incubation, Two Tailed Test

    BRCA1 is required to process Top2–DNA adducts. (A) Mock and BRCA1 immunodepletions from LSS extracts; extracts were blotted with the indicated antibodies. (B) DNA replication in BRCA1-depleted extracts is sensitive to low-dose etoposide. Top: control (ΔMock) and BRCA1-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA; bottom: quantification of three independent experiments. The mean is plotted, and error bars indicate the standard deviation (*, P
    Figure Legend Snippet: BRCA1 is required to process Top2–DNA adducts. (A) Mock and BRCA1 immunodepletions from LSS extracts; extracts were blotted with the indicated antibodies. (B) DNA replication in BRCA1-depleted extracts is sensitive to low-dose etoposide. Top: control (ΔMock) and BRCA1-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA; bottom: quantification of three independent experiments. The mean is plotted, and error bars indicate the standard deviation (*, P

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Standard Deviation

    CtIP regulates resection from Top2 adducts. (A) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated with 0.05 U/µl PflMI or 100 µM etoposide (Etop.). Chromatin was purified at the indicated times and processed for Western blot with Top2, CtIP, RPA70, Ku70, and H3 (loading control) antibodies. NS, no sperm control. (B) Plot of RPA binding as shown in A for control and CtIP-depleted extracts treated with PflMI or etoposide. Shown are mean RPA intensities. Error bars represent SD; n = 3. (C) Chromatin-bound RPA70 was monitored in control (ΔMock) and CtIP-depleted extracts treated with etoposide or ICRF-193. a.u., arbitrary units.
    Figure Legend Snippet: CtIP regulates resection from Top2 adducts. (A) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated with 0.05 U/µl PflMI or 100 µM etoposide (Etop.). Chromatin was purified at the indicated times and processed for Western blot with Top2, CtIP, RPA70, Ku70, and H3 (loading control) antibodies. NS, no sperm control. (B) Plot of RPA binding as shown in A for control and CtIP-depleted extracts treated with PflMI or etoposide. Shown are mean RPA intensities. Error bars represent SD; n = 3. (C) Chromatin-bound RPA70 was monitored in control (ΔMock) and CtIP-depleted extracts treated with etoposide or ICRF-193. a.u., arbitrary units.

    Techniques Used: Incubation, Purification, Western Blot, Recombinase Polymerase Amplification, Binding Assay

    Exo1 cannot process Top2–DNA adducts. (A) Mock, Exo1, or CtIP immunodepletions from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (B) Quantification of Top2–DNA adducts in CtIP-depleted or Exo1-depleted extracts treated with etoposide as described (*, P = 0.02, two-tailed unpaired t test; NS, not statistically significant). (C) Control (ΔMock) or CtIP-depleted replication-incompetent, membrane-free extracts (HSS) were supplemented with sperm nuclei (5,000/µl) and treated with etoposide. (D) Chromatin fractions from extracts in C were blotted with the indicated antibodies.
    Figure Legend Snippet: Exo1 cannot process Top2–DNA adducts. (A) Mock, Exo1, or CtIP immunodepletions from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (B) Quantification of Top2–DNA adducts in CtIP-depleted or Exo1-depleted extracts treated with etoposide as described (*, P = 0.02, two-tailed unpaired t test; NS, not statistically significant). (C) Control (ΔMock) or CtIP-depleted replication-incompetent, membrane-free extracts (HSS) were supplemented with sperm nuclei (5,000/µl) and treated with etoposide. (D) Chromatin fractions from extracts in C were blotted with the indicated antibodies.

    Techniques Used: Two Tailed Test

    Replication of etoposide-treated chromosomal DNA requires CtIP. (A) CtIP immunodepletion from LSS extracts. Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) were probed with the indicated antibodies. (B) Mock and CtIP-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA at the indicated time points. (C) Quantification of three independent experiments as shown in B. The mean is plotted, and error bars indicate the standard deviation. (D) Control (mock) and CtIP-depleted extracts were supplemented with sperm nuclei, and DNA replication of Xenopus sperm nuclei was monitored by alkaline gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA at the indicated time points. (E) DNA replication was monitored as in B in the presence of a low dose (2 µM) of etoposide in mock- or CtIP-depleted extracts, below quantification of three independent experiments (**, P = 0.003, two-tailed unpaired t test; n = 3). (F) Effect of ATM and ATR inhibitors in the sensitivity to low-dose etoposide (2 µM). Bar graph shows quantification of three independent experiments (*, P = 0.014, two-tailed unpaired t test; n = 3).
    Figure Legend Snippet: Replication of etoposide-treated chromosomal DNA requires CtIP. (A) CtIP immunodepletion from LSS extracts. Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) were probed with the indicated antibodies. (B) Mock and CtIP-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA at the indicated time points. (C) Quantification of three independent experiments as shown in B. The mean is plotted, and error bars indicate the standard deviation. (D) Control (mock) and CtIP-depleted extracts were supplemented with sperm nuclei, and DNA replication of Xenopus sperm nuclei was monitored by alkaline gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA at the indicated time points. (E) DNA replication was monitored as in B in the presence of a low dose (2 µM) of etoposide in mock- or CtIP-depleted extracts, below quantification of three independent experiments (**, P = 0.003, two-tailed unpaired t test; n = 3). (F) Effect of ATM and ATR inhibitors in the sensitivity to low-dose etoposide (2 µM). Bar graph shows quantification of three independent experiments (*, P = 0.014, two-tailed unpaired t test; n = 3).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Standard Deviation, Nucleic Acid Electrophoresis, Two Tailed Test

    CtIP–BRCA1 interactions are required for CtIP-dependent processing of etoposide-induced DSBs . (A) Control extract (no damage), extracts treated with etoposide (Etop.), or extracts treated with PflM1 were incubated with the CDK inhibitor roscovitine. Chromatin was isolated at 0 or 40 min and processed for Western blot with the indicated antibodies. Roscovitine inhibits genomic DNA replication (bottom). (B) Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) with or without etoposide were supplemented with sperm nuclei, and replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or CtIP-S328A mutant (xCtIP S328A). Bar graph shows quantification of three independent experiments (*, P = 0.026, two-tailed unpaired t test). (C) Relative quantification of Top2–DNA adducts in CtIP-depleted extracts supplemented with buffer, xCtIP WT, or xCtIP S328A (*, P = 0.035, two-tailed unpaired t test; NS, not statistically significant [P = 0.08]; n = 3). (D) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and treated with either PflM1 endonuclease or etoposide. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or xCtIP S328A. Chromatin was isolated and processed for Western blotting with the indicated antibodies. NS, no sperm control; Ext., extract.
    Figure Legend Snippet: CtIP–BRCA1 interactions are required for CtIP-dependent processing of etoposide-induced DSBs . (A) Control extract (no damage), extracts treated with etoposide (Etop.), or extracts treated with PflM1 were incubated with the CDK inhibitor roscovitine. Chromatin was isolated at 0 or 40 min and processed for Western blot with the indicated antibodies. Roscovitine inhibits genomic DNA replication (bottom). (B) Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) with or without etoposide were supplemented with sperm nuclei, and replication was monitored by agarose gel electrophoresis after incorporation of α-[ 32 P]dCTP into genomic DNA. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or CtIP-S328A mutant (xCtIP S328A). Bar graph shows quantification of three independent experiments (*, P = 0.026, two-tailed unpaired t test). (C) Relative quantification of Top2–DNA adducts in CtIP-depleted extracts supplemented with buffer, xCtIP WT, or xCtIP S328A (*, P = 0.035, two-tailed unpaired t test; NS, not statistically significant [P = 0.08]; n = 3). (D) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and treated with either PflM1 endonuclease or etoposide. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or xCtIP S328A. Chromatin was isolated and processed for Western blotting with the indicated antibodies. NS, no sperm control; Ext., extract.

    Techniques Used: Incubation, Isolation, Western Blot, Agarose Gel Electrophoresis, Recombinant, Mutagenesis, Two Tailed Test

    33) Product Images from "Lowering Etoposide Doses Shifts Cell Demise From Caspase-Dependent to Differentiation and Caspase-3-Independent Apoptosis via DNA Damage Response, Inducing AML Culture Extinction"

    Article Title: Lowering Etoposide Doses Shifts Cell Demise From Caspase-Dependent to Differentiation and Caspase-3-Independent Apoptosis via DNA Damage Response, Inducing AML Culture Extinction

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01307

    0.5 uM etoposide induces nuclear blebbing. Untreated U937 cells (A) and cells treated with 0.5 uM etoposide (B–E) . (C) is the magnification of the area indicated by the square in (B) . ∗ marks nucleoli in transition from ring-shaped nucleolus and nucleolus with nucleolonemas. Results are representative of 2 independent experiments.
    Figure Legend Snippet: 0.5 uM etoposide induces nuclear blebbing. Untreated U937 cells (A) and cells treated with 0.5 uM etoposide (B–E) . (C) is the magnification of the area indicated by the square in (B) . ∗ marks nucleoli in transition from ring-shaped nucleolus and nucleolus with nucleolonemas. Results are representative of 2 independent experiments.

    Techniques Used:

    TEM analysis of cells induced to apoptosis by etoposide. (A) U937 treated with 50 uM etoposide showing canonical apoptotic figures, including chromatin condensation at the nuclear margin. (B–D) Cells treated with 0.5 uM etoposide. (B) shows recognizable apoptotic figures, but with atypical membrane features, e.g., two nuclear fragments juxtaposed and separated by pore-enriched double nuclear membrane (white arrow) or interruptions of the nuclear membrane (arrowhead). ∗ indicates nucleolar alterations. (C) Condensed chromatin spherical masses without nuclear membrane (arrowhead). (D) Fourfold membrane network connecting dense chromatin masses in late apoptosis, with the outer sheet wrapped by ribosomes (arrows).
    Figure Legend Snippet: TEM analysis of cells induced to apoptosis by etoposide. (A) U937 treated with 50 uM etoposide showing canonical apoptotic figures, including chromatin condensation at the nuclear margin. (B–D) Cells treated with 0.5 uM etoposide. (B) shows recognizable apoptotic figures, but with atypical membrane features, e.g., two nuclear fragments juxtaposed and separated by pore-enriched double nuclear membrane (white arrow) or interruptions of the nuclear membrane (arrowhead). ∗ indicates nucleolar alterations. (C) Condensed chromatin spherical masses without nuclear membrane (arrowhead). (D) Fourfold membrane network connecting dense chromatin masses in late apoptosis, with the outer sheet wrapped by ribosomes (arrows).

    Techniques Used: Transmission Electron Microscopy

    0.5 uM etoposide promotes intracellular granularity in U937 cells. (A) Cytofluorimetric dot plot analysis of forward (size) vs. side (granularity) scatter in U937 cells untreated or after 24 or 48 h of 0.5 uM etoposide, before (top line) or at 30 min after addition of 200 ng/mL PMA (bottom line). Representative histogram of 3 experiments performed with similar results; the % values indicate the fraction of cells falling in the gated area. (B) Cytofluorimetric analysis of superoxide production (DHE signal) in U937 untreated or after 24–48 h of 0.5 uM etoposide, before (left bars) or at 30 min after addition of 200 ng/mL PMA (right bars). Results are provided as the mean value, and are the average of 3 independent measurements ± SD: ∗ p
    Figure Legend Snippet: 0.5 uM etoposide promotes intracellular granularity in U937 cells. (A) Cytofluorimetric dot plot analysis of forward (size) vs. side (granularity) scatter in U937 cells untreated or after 24 or 48 h of 0.5 uM etoposide, before (top line) or at 30 min after addition of 200 ng/mL PMA (bottom line). Representative histogram of 3 experiments performed with similar results; the % values indicate the fraction of cells falling in the gated area. (B) Cytofluorimetric analysis of superoxide production (DHE signal) in U937 untreated or after 24–48 h of 0.5 uM etoposide, before (left bars) or at 30 min after addition of 200 ng/mL PMA (right bars). Results are provided as the mean value, and are the average of 3 independent measurements ± SD: ∗ p

    Techniques Used:

    0.5 uM etoposide promotes granulocytic-like morphology and differentiation-related apoptosis. (A) Cells with peculiar fragmentation of the nuclei in 2–3 segments with irregular shape, strongly resembling polymorphonuclear cell nuclei. Nuclei show dilated perinuclear membrane blank (arrowhead). (B) Cells presenting apoptotic nuclei and granulocyte-like characteristics, including electrondense granules at the cell periphery or in the process of being extruded.
    Figure Legend Snippet: 0.5 uM etoposide promotes granulocytic-like morphology and differentiation-related apoptosis. (A) Cells with peculiar fragmentation of the nuclei in 2–3 segments with irregular shape, strongly resembling polymorphonuclear cell nuclei. Nuclei show dilated perinuclear membrane blank (arrowhead). (B) Cells presenting apoptotic nuclei and granulocyte-like characteristics, including electrondense granules at the cell periphery or in the process of being extruded.

    Techniques Used:

    0.5 uM etoposide induces a caffeine-sensitive DNA damage response determining apoptosis and differentiation. (A) Cell cycle analysis of U937 cells treated with 0.5 uM etoposide ± caffeine (caff). Representative histograms of 4 experiments performed with similar results. (B) Apoptosis (nuclear fragmentation) in U937 cells treated with 50 uM or 0.5 uM etoposide ± caffeine (caff). Results are representative of 3 independent experiments ± SD: ∗ p
    Figure Legend Snippet: 0.5 uM etoposide induces a caffeine-sensitive DNA damage response determining apoptosis and differentiation. (A) Cell cycle analysis of U937 cells treated with 0.5 uM etoposide ± caffeine (caff). Representative histograms of 4 experiments performed with similar results. (B) Apoptosis (nuclear fragmentation) in U937 cells treated with 50 uM or 0.5 uM etoposide ± caffeine (caff). Results are representative of 3 independent experiments ± SD: ∗ p

    Techniques Used: Cell Cycle Assay

    0.5 uM etoposide induces caspase-independent apoptosis. (A) Fraction of apoptotic U937 cells after treatment with 50 uM or 0.5 uM etoposide ± pan caspase inhibitor (Z-VAD-fmk). Effect of caspase-3 (B) and caspase-2 (C) inhibitors on 50 uM and 0.5 uM etoposide-induced apoptosis (measured as nuclear fragmentation). Results are the average of 3 independent measurements ± SD: ∗ p
    Figure Legend Snippet: 0.5 uM etoposide induces caspase-independent apoptosis. (A) Fraction of apoptotic U937 cells after treatment with 50 uM or 0.5 uM etoposide ± pan caspase inhibitor (Z-VAD-fmk). Effect of caspase-3 (B) and caspase-2 (C) inhibitors on 50 uM and 0.5 uM etoposide-induced apoptosis (measured as nuclear fragmentation). Results are the average of 3 independent measurements ± SD: ∗ p

    Techniques Used:

    34) Product Images from "Differential Cytotoxic Activity of a Novel Palladium-Based Compound on Prostate Cell Lines, Primary Prostate Epithelial Cells and Prostate Stem Cells"

    Article Title: Differential Cytotoxic Activity of a Novel Palladium-Based Compound on Prostate Cell Lines, Primary Prostate Epithelial Cells and Prostate Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064278

    Chemical structures. (A) Palladium complex [PdCl(terpy)](sac)·2 H 2 O, (sac = saccharinate, and terpy = 2,2′:6′,2″-terpyridine). M = Palladium(II) (B) Cisplatin (C) Etoposide.
    Figure Legend Snippet: Chemical structures. (A) Palladium complex [PdCl(terpy)](sac)·2 H 2 O, (sac = saccharinate, and terpy = 2,2′:6′,2″-terpyridine). M = Palladium(II) (B) Cisplatin (C) Etoposide.

    Techniques Used:

    DNA-damaging effect and effect on Cell Cycle Status of Pd complex. (A) Primary cultures isolated from two patients with prostate carcinoma were assessed. Cells were stained and scored for nuclear foci indicative of DNA damage Representative examples of cells negative and positive for nuclear foci are shown. (B) Normal (PNT2-C2) and benign (BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) and (C) four primary cultures derived from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or cisplatin as control treatments. Cell cycle phase was measured using propidium iodide staining and flow cytometry analysis.
    Figure Legend Snippet: DNA-damaging effect and effect on Cell Cycle Status of Pd complex. (A) Primary cultures isolated from two patients with prostate carcinoma were assessed. Cells were stained and scored for nuclear foci indicative of DNA damage Representative examples of cells negative and positive for nuclear foci are shown. (B) Normal (PNT2-C2) and benign (BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) and (C) four primary cultures derived from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or cisplatin as control treatments. Cell cycle phase was measured using propidium iodide staining and flow cytometry analysis.

    Techniques Used: Isolation, Staining, Derivative Assay, Flow Cytometry, Cytometry

    Expression of apoptosis and autophagy-related proteins following treatment with Pd complex. Normal (PNT2-C2) and benign (BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) (A) and four primary cultures (B) one derived from patient with BPH and three derived from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or cisplatin as control treatments. Lysates were harvested and Western blotting was carried out staining for cleaved caspase-3 indicative of apoptosis induction or LC3B protein, indicative of autophagy. (C) Images of primary cells (sample 23912) treated with Pd complex and stained with LC3-B antibody. Shown are three example images of untreated cells and cells treated with 12 µM and 25 µM Pd complex. Autophagosome vesicles are clearly visible in red.
    Figure Legend Snippet: Expression of apoptosis and autophagy-related proteins following treatment with Pd complex. Normal (PNT2-C2) and benign (BPH-1) cell lines, three cancer cell lines (P4E6, PC-3 and LNCaP) (A) and four primary cultures (B) one derived from patient with BPH and three derived from patients with prostate carcinoma were treated with three concentrations of palladium complex or etoposide or cisplatin as control treatments. Lysates were harvested and Western blotting was carried out staining for cleaved caspase-3 indicative of apoptosis induction or LC3B protein, indicative of autophagy. (C) Images of primary cells (sample 23912) treated with Pd complex and stained with LC3-B antibody. Shown are three example images of untreated cells and cells treated with 12 µM and 25 µM Pd complex. Autophagosome vesicles are clearly visible in red.

    Techniques Used: Expressing, Derivative Assay, Western Blot, Staining

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

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

    Journal: Genes & Development

    doi: 10.1101/gad.1047003

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

    Techniques Used: TUNEL Assay

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

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

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

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

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

    Techniques Used: Western Blot

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

    Techniques Used: Protease Inhibitor

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

    Techniques Used: Protease Inhibitor, Staining, FACS

    36) Product Images from "Evaluation of pemetrexed and etoposide as therapeutic regimens for human papillomavirus-positive oral and oropharyngeal cancer"

    Article Title: Evaluation of pemetrexed and etoposide as therapeutic regimens for human papillomavirus-positive oral and oropharyngeal cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200509

    Cisplatin/etoposide or cisplatin/pemetrexed combination is more effectively in HPV positive HNSCC cell lines. (A) MTT assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 1, 5, and 10 μM etoposide. (B) MTT assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 1, 5, and 10 μM pemetrexed. (C) TUNEL assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 10 μM pemetrexed. p values: **
    Figure Legend Snippet: Cisplatin/etoposide or cisplatin/pemetrexed combination is more effectively in HPV positive HNSCC cell lines. (A) MTT assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 1, 5, and 10 μM etoposide. (B) MTT assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 1, 5, and 10 μM pemetrexed. (C) TUNEL assay results in UN-SCC-1 and 93-VU-147T cells treated with 1 μM cisplatin and 10 μM pemetrexed. p values: **

    Techniques Used: MTT Assay, TUNEL Assay

    Cytotoxicity of etoposide according to HPV status in oral cancer cell lines. (A, B) MTT assay results in Ho-1-N-1 and YD10B cells treated with 0, 1, 5, and 10 μM of etoposide after the transfection of Lenti-blank or Lenti-E6/7. (C, D) LDH assay result in Ho-1-N-1 and YD10B cells, representative of HPV negative and HPV positive cell lines, were treated with increasing concentrations of etoposide. p values: *
    Figure Legend Snippet: Cytotoxicity of etoposide according to HPV status in oral cancer cell lines. (A, B) MTT assay results in Ho-1-N-1 and YD10B cells treated with 0, 1, 5, and 10 μM of etoposide after the transfection of Lenti-blank or Lenti-E6/7. (C, D) LDH assay result in Ho-1-N-1 and YD10B cells, representative of HPV negative and HPV positive cell lines, were treated with increasing concentrations of etoposide. p values: *

    Techniques Used: MTT Assay, Transfection, Lactate Dehydrogenase Assay

    Cytotoxicity of cisplatin/etoposide or cisplatin/pemetrexed combination regimens according to HPV status in oral cancer cell lines. (A, B) MTT assay results in Ho-1-N-1 and YD10B cells treated with 1 μM cisplatin and 0, 1, 5, and 10 μM etoposide, according to HPV status. (C, D) MTT assay results in Ho-1-N-1 and YD10B cells treated with 1 μM cisplatin and 0, 1, 5, and 10 μM pemetrexed, according to HPV status. p values: **
    Figure Legend Snippet: Cytotoxicity of cisplatin/etoposide or cisplatin/pemetrexed combination regimens according to HPV status in oral cancer cell lines. (A, B) MTT assay results in Ho-1-N-1 and YD10B cells treated with 1 μM cisplatin and 0, 1, 5, and 10 μM etoposide, according to HPV status. (C, D) MTT assay results in Ho-1-N-1 and YD10B cells treated with 1 μM cisplatin and 0, 1, 5, and 10 μM pemetrexed, according to HPV status. p values: **

    Techniques Used: MTT Assay

    37) Product Images from "Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line"

    Article Title: Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2015.1

    Downregulation of Rap1 eliminated the inhibition effects of TRF2 on etoposide-induced ATM activation. ( a ) TRF2 eukaryotic expression vector was transfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. ( b ) Rap1 siRNA vector and TRF2 eukaryotic expression vector were cotransfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. ( c ) Rap1 siRNA vector was transfected into SGC7901/VCR. Expression of TRF2, Rap1, ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot.
    Figure Legend Snippet: Downregulation of Rap1 eliminated the inhibition effects of TRF2 on etoposide-induced ATM activation. ( a ) TRF2 eukaryotic expression vector was transfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. ( b ) Rap1 siRNA vector and TRF2 eukaryotic expression vector were cotransfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. ( c ) Rap1 siRNA vector was transfected into SGC7901/VCR. Expression of TRF2, Rap1, ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot.

    Techniques Used: Inhibition, Activation Assay, Expressing, Plasmid Preparation, Transfection, Western Blot

    TRF2 and Rap1 upregulation induced by etoposide in gastric cancer cells. ( a ) Protein expression levels of TRF2 and Rap1 detected by western blot in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. ( b ) Relative mRNA expression levels of TRF2 and Rap1 detected by real-time PCR analysis in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. * P
    Figure Legend Snippet: TRF2 and Rap1 upregulation induced by etoposide in gastric cancer cells. ( a ) Protein expression levels of TRF2 and Rap1 detected by western blot in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. ( b ) Relative mRNA expression levels of TRF2 and Rap1 detected by real-time PCR analysis in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Rap1 is involved in TRF2-mediated etoposide resistance in gastric cancer cells. ( a ) Rap1 siRNA vectors were transfected into SGC7901/VCR, respectively. Rap1 expression after treated with 20 μg/ml of etoposide for 6 h was detected by western blot and real-time PCR analysis. * P
    Figure Legend Snippet: Rap1 is involved in TRF2-mediated etoposide resistance in gastric cancer cells. ( a ) Rap1 siRNA vectors were transfected into SGC7901/VCR, respectively. Rap1 expression after treated with 20 μg/ml of etoposide for 6 h was detected by western blot and real-time PCR analysis. * P

    Techniques Used: Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    38) Product Images from "Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells"

    Article Title: Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells

    Journal: Archives of Toxicology

    doi: 10.1007/s00204-018-2170-7

    Etoposide upregulates apoptosis signaling in hiPSC-CMs. a Fluorescent images showing live cells (blue), apoptotic cells (green), and necrotic cells (red) in hiPSC-CMs after ETP treatment for 48 h. Scale bar represents 10 µm. b, c hiPSC-CMs were co-treated with ETP and 10 µM Pifithrin-α for 48 h. Real-time data of hPSC-CMs cell index and beating rate were obtained using xCELLigence RTCA system. Representative graphs display normalized CI and % beating rate values, respectively, showing Pifithrin-α had significant effect in preventing ETP-induced cardiotoxicity ( n = 3, error bars represent ± SEM) ( t test, * p ≤ 0.05, *** p ≤ 0.001) (see also Fig. S2A). (Color figure online)
    Figure Legend Snippet: Etoposide upregulates apoptosis signaling in hiPSC-CMs. a Fluorescent images showing live cells (blue), apoptotic cells (green), and necrotic cells (red) in hiPSC-CMs after ETP treatment for 48 h. Scale bar represents 10 µm. b, c hiPSC-CMs were co-treated with ETP and 10 µM Pifithrin-α for 48 h. Real-time data of hPSC-CMs cell index and beating rate were obtained using xCELLigence RTCA system. Representative graphs display normalized CI and % beating rate values, respectively, showing Pifithrin-α had significant effect in preventing ETP-induced cardiotoxicity ( n = 3, error bars represent ± SEM) ( t test, * p ≤ 0.05, *** p ≤ 0.001) (see also Fig. S2A). (Color figure online)

    Techniques Used:

    Etoposide induces mitochondrial damage, increased ROS production and loss of mitochondrial membrane potential ( m ∆ ψ ) in hiPSC-CMs. a TEM images showing morphological alterations in mitochondrial membrane and cristae structures in hiPSC-CMs treated with ETP compared to untreated CMs. Scale bar represents 500 nm. b Fluorescent images showing increase DHE staining in hiPSC-CMs treated with ETP compared to untreated CMs, indicating net increase in ROS production. Nuclei are stained with DAPI which also show increased nuclear size after ETP treatment. Scale bar represents 20 µm. c Determination of mitochondrial membrane potential through JC-1 staining. Mitochondria of hiPSC-CMs after incubation with JC-1 dye, illustrating the heterogeneity in mitochondrial membrane potential in the same cell. The mitochondria membrane potential was found to be interrupted after DOX (positive control) and ETP treatment, as evidenced by reduction in the JC-1 red fluorescence signal. In addition hPSC-CMs treated with 30 µm ETP for 48 h showed increased mitochondrial fragmentation further supporting the TEM data. Scale bar represents 20 µm (upper panel), 5 µm (lower panel)
    Figure Legend Snippet: Etoposide induces mitochondrial damage, increased ROS production and loss of mitochondrial membrane potential ( m ∆ ψ ) in hiPSC-CMs. a TEM images showing morphological alterations in mitochondrial membrane and cristae structures in hiPSC-CMs treated with ETP compared to untreated CMs. Scale bar represents 500 nm. b Fluorescent images showing increase DHE staining in hiPSC-CMs treated with ETP compared to untreated CMs, indicating net increase in ROS production. Nuclei are stained with DAPI which also show increased nuclear size after ETP treatment. Scale bar represents 20 µm. c Determination of mitochondrial membrane potential through JC-1 staining. Mitochondria of hiPSC-CMs after incubation with JC-1 dye, illustrating the heterogeneity in mitochondrial membrane potential in the same cell. The mitochondria membrane potential was found to be interrupted after DOX (positive control) and ETP treatment, as evidenced by reduction in the JC-1 red fluorescence signal. In addition hPSC-CMs treated with 30 µm ETP for 48 h showed increased mitochondrial fragmentation further supporting the TEM data. Scale bar represents 20 µm (upper panel), 5 µm (lower panel)

    Techniques Used: Transmission Electron Microscopy, Staining, Incubation, Positive Control, Fluorescence

    Liproxstatin-1 improves cardiomyocytes functional properties after etoposide treatment. The hiPSC-CMs were co-treated with ETP and 200 nM Liproxstatin-1 (ferroptosis inhibitor) for 48 h. Real-time data of hPSC-CMs cell index, beating rate, and beating amplitude were obtained using xCELLigence RTCA system. Representative graphs display a normalized CI and b % beating rate values, respectively, showing even though Liproxstatin-1 had no significant effect in preventing ETP-induced cytotoxicity; it significantly improved hPSC-CMs ability to recover from ETP-induced alterations in beating rate and beating amplitude ( n = 3, error bars represent ± SEM) ( t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) (see also Fig. S3A)
    Figure Legend Snippet: Liproxstatin-1 improves cardiomyocytes functional properties after etoposide treatment. The hiPSC-CMs were co-treated with ETP and 200 nM Liproxstatin-1 (ferroptosis inhibitor) for 48 h. Real-time data of hPSC-CMs cell index, beating rate, and beating amplitude were obtained using xCELLigence RTCA system. Representative graphs display a normalized CI and b % beating rate values, respectively, showing even though Liproxstatin-1 had no significant effect in preventing ETP-induced cytotoxicity; it significantly improved hPSC-CMs ability to recover from ETP-induced alterations in beating rate and beating amplitude ( n = 3, error bars represent ± SEM) ( t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) (see also Fig. S3A)

    Techniques Used: Functional Assay

    Single high dose of etoposide induces arrhythmic beating and cytotoxicity in hiPSC-CMs. a Schematic representation and experimental setup of the in vitro cardiotoxicity test model. For functional studies, the synchronously beating hiPSC-CMs in the E-plate Cardio 96 were exposed to ETP (single high-dose exposure) for 48 h. After exposure, the ETP was washed out and the cells were further incubated for 48 h. The effects of ETP on hPSC-CMs functional characteristics were monitored by the xCELLigence RTCA Cardio system. For qRT-PCR studies, RNA from ETP-treated and untreated control cells were harvested at day 2. b – e Functional studies of ETP-treated hiPSC-CMs. The representative graphs display, b normalized CI values showing ETP-induced cytotoxicity ( n = 3, error bars represent ± SEM), c % beating rate alterations induced by single dose of ETP in hiPSC-CMs ( n = 3, error bars represent ± SEM) ( t test, * p
    Figure Legend Snippet: Single high dose of etoposide induces arrhythmic beating and cytotoxicity in hiPSC-CMs. a Schematic representation and experimental setup of the in vitro cardiotoxicity test model. For functional studies, the synchronously beating hiPSC-CMs in the E-plate Cardio 96 were exposed to ETP (single high-dose exposure) for 48 h. After exposure, the ETP was washed out and the cells were further incubated for 48 h. The effects of ETP on hPSC-CMs functional characteristics were monitored by the xCELLigence RTCA Cardio system. For qRT-PCR studies, RNA from ETP-treated and untreated control cells were harvested at day 2. b – e Functional studies of ETP-treated hiPSC-CMs. The representative graphs display, b normalized CI values showing ETP-induced cytotoxicity ( n = 3, error bars represent ± SEM), c % beating rate alterations induced by single dose of ETP in hiPSC-CMs ( n = 3, error bars represent ± SEM) ( t test, * p

    Techniques Used: In Vitro, Functional Assay, Incubation, Quantitative RT-PCR

    Etoposide causes alterations in calcium handling in hiPSC-CMs. a Confocal line-scan images showing changes in intracellular [Ca 2+ ] i in a Rhod-2, AM loaded hiPSC-CM. The images show alterations in spontaneous whole-cell Ca 2+ transients in response to ETP treatment (upper panel). Scale bar represents, time − 1 s and distance − 10 µm. Representative tracings of spontaneous Ca 2+ transients (black arrow head) in hiPSC-CMs from untreated and ETP-treated groups (lower panel). b Graphs representing Ca 2+ transient parameters measured from hiPSC-CMs treated with ETP. F / F 0 , Ca 2+ transient amplitude where F 0 is the averaged background-corrected resting fluorescence intensity; TTP, time-to-peak; T90%, 90% recovery of F max ; [Δ F /Δ T ] max , the maximum steepness; FWHM, full-width at half-maximum. ( n = 25, error bars represent ± SEM) ( t test, * p ≤ 0.05, ** p ≤ 0.01)
    Figure Legend Snippet: Etoposide causes alterations in calcium handling in hiPSC-CMs. a Confocal line-scan images showing changes in intracellular [Ca 2+ ] i in a Rhod-2, AM loaded hiPSC-CM. The images show alterations in spontaneous whole-cell Ca 2+ transients in response to ETP treatment (upper panel). Scale bar represents, time − 1 s and distance − 10 µm. Representative tracings of spontaneous Ca 2+ transients (black arrow head) in hiPSC-CMs from untreated and ETP-treated groups (lower panel). b Graphs representing Ca 2+ transient parameters measured from hiPSC-CMs treated with ETP. F / F 0 , Ca 2+ transient amplitude where F 0 is the averaged background-corrected resting fluorescence intensity; TTP, time-to-peak; T90%, 90% recovery of F max ; [Δ F /Δ T ] max , the maximum steepness; FWHM, full-width at half-maximum. ( n = 25, error bars represent ± SEM) ( t test, * p ≤ 0.05, ** p ≤ 0.01)

    Techniques Used: Fluorescence

    Etoposide induces cytoskeletal disorganization in hiPSC-CMs. a, b Immunostaining of cardiac sarcomeric α-actinin (α-Actinin) and cardiac troponin T (cTnT) in untreated and ETP-treated hiPSC-CMs. Nuclei are stained with DAPI. Scale bar represents 50, 5 µm. c Represents TEM images of untreated and ETP-treated hiPSC-CMs. Scale bar represents 5000 nm. (1, Z-line; 2, sarcomere; 3, mitochondria; 4, myofibril bundles)
    Figure Legend Snippet: Etoposide induces cytoskeletal disorganization in hiPSC-CMs. a, b Immunostaining of cardiac sarcomeric α-actinin (α-Actinin) and cardiac troponin T (cTnT) in untreated and ETP-treated hiPSC-CMs. Nuclei are stained with DAPI. Scale bar represents 50, 5 µm. c Represents TEM images of untreated and ETP-treated hiPSC-CMs. Scale bar represents 5000 nm. (1, Z-line; 2, sarcomere; 3, mitochondria; 4, myofibril bundles)

    Techniques Used: Immunostaining, Staining, Transmission Electron Microscopy

    39) Product Images from "ZNF185 is a p53 target gene following DNA damage"

    Article Title: ZNF185 is a p53 target gene following DNA damage

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101639

    ZNF185 is up-regulated upon DNA damage. ( A ) qPCR analysis of ZNF185 and CDKN1A mRNA levels in HCT116 after 25 µM etoposide treatment. ** P
    Figure Legend Snippet: ZNF185 is up-regulated upon DNA damage. ( A ) qPCR analysis of ZNF185 and CDKN1A mRNA levels in HCT116 after 25 µM etoposide treatment. ** P

    Techniques Used: Real-time Polymerase Chain Reaction

    ZNF185 is involved in the cytoskeleton remodelling upon DNA damage. ( A ) FACS analysis of cell cycle content of the Ker-CT treated with either DMSO or 100 µM etoposide for 24 h after ZNF185 knock-down with two different siRNAs. Western blot confirms ZNF185 silencing. ( B ) EdU-incorporation assay by FACS showing % of EdU-positive Ker-CT shZNF185 cells. Western blot confirms the ZNF185 knock-down. ( C ) Immunofluorescence analysis of ZNF185 expression in Ker-CT treated with either DMSO or 100 µM etoposide for 16 h. Phalloidin was used for cytoskeleton staining. Scale bar: 50 µm. ( D ) Immunofluorescence analysis of vinculin distribution in Ker-CT treated with either DMSO or 100 µM etoposide and knocked-down for ZNF185. Phalloidin was used for cytoskeleton staining. Scale bar: 20 µm. In the right panel is shown the quantification of % of polarized cells in ten random fields. Western blot shows ZNF185, p53, and p21 levels.
    Figure Legend Snippet: ZNF185 is involved in the cytoskeleton remodelling upon DNA damage. ( A ) FACS analysis of cell cycle content of the Ker-CT treated with either DMSO or 100 µM etoposide for 24 h after ZNF185 knock-down with two different siRNAs. Western blot confirms ZNF185 silencing. ( B ) EdU-incorporation assay by FACS showing % of EdU-positive Ker-CT shZNF185 cells. Western blot confirms the ZNF185 knock-down. ( C ) Immunofluorescence analysis of ZNF185 expression in Ker-CT treated with either DMSO or 100 µM etoposide for 16 h. Phalloidin was used for cytoskeleton staining. Scale bar: 50 µm. ( D ) Immunofluorescence analysis of vinculin distribution in Ker-CT treated with either DMSO or 100 µM etoposide and knocked-down for ZNF185. Phalloidin was used for cytoskeleton staining. Scale bar: 20 µm. In the right panel is shown the quantification of % of polarized cells in ten random fields. Western blot shows ZNF185, p53, and p21 levels.

    Techniques Used: FACS, Western Blot, Immunofluorescence, Expressing, Staining

    40) Product Images from "Depletion of SMC5/6 sensitizes male germ cells to DNA damage"

    Article Title: Depletion of SMC5/6 sensitizes male germ cells to DNA damage

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-07-0459

    Conditional mutation of Smc5 results in increased sensitivity to etoposide, producing a similar phenotype seen for irradiation. (A, B) Periodic acid–Schiff stained tubule cross-sections of adult control and Smc5 cKO ( Stra8-Cre ) testes extracted at 3, 5, or 8 d postexposure to etoposide. Smc5 cKO testes have increased numbers of abnormal enlarged round spermatids (*) at 8 d postexposure. Roman numerals correspond to the seminiferous tubule stage. Mice were ≥8 wk old. Scale bar: 20 μm.
    Figure Legend Snippet: Conditional mutation of Smc5 results in increased sensitivity to etoposide, producing a similar phenotype seen for irradiation. (A, B) Periodic acid–Schiff stained tubule cross-sections of adult control and Smc5 cKO ( Stra8-Cre ) testes extracted at 3, 5, or 8 d postexposure to etoposide. Smc5 cKO testes have increased numbers of abnormal enlarged round spermatids (*) at 8 d postexposure. Roman numerals correspond to the seminiferous tubule stage. Mice were ≥8 wk old. Scale bar: 20 μm.

    Techniques Used: Mutagenesis, Irradiation, Staining, Mouse Assay

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    Millipore etoposide
    Role of Egr-1 in <t>etoposide-induced</t> BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).
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    Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Role of Egr-1 in etoposide-induced BRCA1 expression. (A) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total RNA was isolated and Egr-1 mRNA expression was assessed by Northern blotting with 32 P-labeled Egr-1 cDNA. The same blot was re-probed with 32 P-labeled GAPDH cDNA as an internal control. (B) Serum-starved HeLa cells were treated with 100 μM etoposide for different time periods. Total cell lysates were prepared and subjected to Western blot analysis with rabbit anti-Egr-1 antibody. The same blot was reprobed with anti-GAPDH antibody as an internal control. (C) HeLa cells were transiently co-transfected with 0.5 μg pBRCA1-Luc(–1066/+135) and an shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ), along with 50 ng of the pRL-null vector plasmid. After 24 h, the cells were left untreated or treated with 100 μM etoposide for 8 h, and the luciferase activity was measured. Egr-1 is indicated by an arrow. The knockdown of Egr-1 expression was verified by Western blot analysis ( upper panel ). Luciferase activity is shown as the mean ± SD of three independent experiments performed in triplicate (bottom graph). **P < 0.01. (D) HeLa cells were transiently transfected with 0.5 μg shRNA plasmid, pSilencer/scrambled (control siRNA; Cont ) or pSilencer/siEgr1 ( Egr-1 ). After 24 h, the cells were left untreated or treated with 100 μM etoposide for 3 h. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against Egr-1 and BRCA1. Egr-1 is indicated by an arrow. The same blot was reprobed with anti-GAPDH antibody as an internal control. The relative band intensities were measured by quantitative scanning densitometer ( bottom graph ).

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Isolation, Northern Blot, Labeling, Western Blot, Transfection, shRNA, Plasmid Preparation, Luciferase, Activity Assay

    Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Egr-1 transactivates the BRCA1 promoter through direct binding to the EBS. (A) HeLa cells were co-transfected with 0.5 μg wild-type (WT) p BRCA1 -Luc(–1066/+135) and different concentrations of the empty vector (pcDNA3.1zeo) or Egr-1 expression plasmid (pcDNA3.1zeo/Egr1), as indicated. After 48 h, the cells were collected and analyzed for luciferase activity. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. (B and C) Purified recombinant Egr-1 protein (B) and nuclear extracts from HeLa cells treated with 100 μM etoposide for 1 h (C) were incubated with 32 P-labeled oligodeoxynucleotide probes that contain EBS. For competition, unlabeled oligodeoxynucleotides (Competitor) were added at 10-fold or 100-fold excess. Arrowheads indicate DNA-Egr-1 complexes.

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Purification, Recombinant, Incubation, Labeling

    Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.

    Journal: BMB Reports

    Article Title: Egr-1 regulates the transcription of the BRCA1 gene by etoposide

    doi: 10.5483/BMBRep.2013.46.2.202

    Figure Lengend Snippet: Effect of etoposide on the induction of BRCA1 expression. (A) HeLa cells were treated with 100 μM etoposide for different time periods. Whole cell extracts were prepared and subjected to Western blotting with antibodies directed against BRCA1, p53 and p21. The ∼220-kDa full length and ∼90-kDa fragment of BRCA1 are indicated by an arrow and an arrowhead, respectively. The same blot was reprobed with anti-GAPDH antibody as an internal control. The blots shown are representative of the results obtained from three independent experiments. (B) Total RNA was isolated and the levels of BRCA1 mRNA were measured by QRT-PCR. Relative levels are normalized to the level of gapdh mRNA. The data shown represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, compared with the untreated control cells. (C) HeLa cells grown in 12-well plates were transfected with 0.5 μg of the BRCA1 promoter reporter plasmid, p BRCA1 -Luc(–1066/+135), along with 50 ng of the pRL-null vector. After 24 h, the cells were either untreated or treated with 50 μM or 100 μM etoposide for 8 h. The firefly luciferase activity was normalized to the Renilla activity. The data shown represent the mean ± SD of three independent experiments performed in triplicate. *P < 0.05; **P < 0.01, compared with the untreated control cells.

    Article Snippet: Etoposide was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Western Blot, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The M 3 -muscarinic-receptor-mediated anti-apoptotic response and transcriptional activation are both attenuated by actinomycin D treatment ( A ) CHO-M 3 cells seeded on to 6-well plates were serum-starved for 2 h before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for the indicated times. [ 3 H]UTP incorporation was determined by scintillation counting. Data shown is the agonist-induced incorporation of [ 3 H]UTP subtracted from that observed in the absence of agonist, and represent the means±S.E.M. ( n =3). ( B ) CHO-M 3 cells seeded on 6-well plates were serum-starved for 2 h and then incubated with or without actinomycin D (100 ng/ml) for 30 min before the addition of [ 3 H]UTP (1 μCi/ml). Cells were then treated with carbachol (1 mM) for 30 min. [ 3 H]UTP incorporation was determined by scintillation counting. Results are means±S.E.M. ( n =3). ( C ) CHO-M 3 cells seeded on to 10 cm 2 plates were pre-incubated with or without actinomycin D (100 ng/ml) for 30 min. Cells were then treated with carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h and cell lysates processed for caspase activity. Caspase activity of 100% is given as the increase in caspase activity in the presence of etoposide. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Incubation, Activity Assay

    Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Decreased mRNA levels of Bcl-2 following etoposide treatment could not be inhibited by muscarinic receptor stimulation in CHO-M 3 cells CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with vehicle or etoposide (250 μM) for 16 h in the absence or presence of carbachol (1 mM). Total RNA was then extracted and reverse transcribed. RT-PCR using primers specific for Bcl-2 and the internal control β-actin was performed using SYBR® Green mastermix. Levels of the Bcl-2 transcript were then normalized to β-actin and expressed as a percentage of the control vehicle-treated cells. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay

    The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The decrease in the expression levels of the anti-apoptotic Bcl-2 protein following incubation of cells with etoposide can be inhibited by activation of the M 3 -muscarinic receptor CHO-M 3 cells seeded on to 10 cm 2 plates were incubated with or without carbachol (CCh; 1mM) either for 2 min before a 16-h treatment with etoposide (250 μM) or incubated with etoposide and carbachol for 16 h. In the case of cells treated with carbachol for 2 min, the carbachol stimulation was stopped by the addition of the muscarinic receptor antagonist atropine (0.5 μM) followed by three washes in α-MEM before being incubated with etoposide for 16 h. Cell lysates were then prepared and separated by SDS/PAGE (12% gels; 100 μg of protein/well). The gel was then probed for Bcl-2 immunoreactivity. The nitrocellulose was then stripped and re-probed with ERK antisera in order to check for equal lane loading (right-hand panels). Gels are representative of three separate experiments.

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Expressing, Incubation, Activation Assay, SDS Page

    The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: The M 3 -muscarinic receptor anti-apoptotic response is independent of extracellular calcium CHO-M 3 cells were seeded on 10 cm 2 plates. Cells were incubated in Krebs buffer (+calcium) or in Krebs buffer lacking calcium and supplemented with 3 mM EDTA (−calcium) for 3 min. CHO-M 3 cells were treated with carbachol (CCh; 1 mM) for 5 min and cells were washed three times with α-MEM. CHO-M 3 cells were then incubated for 16 h with etoposide (250 μM) and cell lysates processed for caspase activity. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Activity Assay

    Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Both M 3 -muscarinic and vasopressin V 1 A receptors increase inositol phosphate production, but only the M 3 -muscarinic receptor mediates an anti-apoptotic response ( A ) CHO-M 3 and CHO-V 1 A cells plated on 24-well plates were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) for 25 min. The reactions were terminated with 1 M trichloroacetic acid and inositol phosphate production was determined as described in the Materials and methods section. ( B ) CHO-M 3 and CHO-V 1 A cells were stimulated with carbachol (CCh; 1 mM) or vasopressin (1 μM) and etoposide (250 μM). Cells were incubated for 16 h at 37 °C. Floating and attached cells were harvested and pooled, and cell lysates were processed for caspase activity as described in the Materials and methods section. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Incubation, Activity Assay

    Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Time-course of etoposide-induced decrease in Bcl-2 levels and caspase activation CHO-M 3 cells seeded on 10 cm 2 plates were incubated with etoposide (250 μM) for the times indicated. Cell lysates were prepared and run on a SDS/PAGE (12% gels) and Western blotted for Bcl-2. Data shown is representative of three separate experiments (upper panel). In parallel experiments, a time course for caspase activity was determined following etoposide (Et; 250 μM) treatment for the indicated times. Results for caspase activation are means±S.E.M. ( n =3) (lower panel).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Activity Assay

    Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).

    Journal: Biochemical Journal

    Article Title: Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway

    doi: 10.1042/BJ20031705

    Figure Lengend Snippet: Attenuation of etoposide-mediated caspase activation by M 3 -muscarinic receptors in CHO-M 3 and SHSY-5Y cells ( A ) CHO-M 2 and CHO-M 3 cells seeded on 10 cm 2 plates were treated with vehicle (Control), carbachol (CCh; 1 mM) and/or etoposide (250 μM) for 16 h, and cell lysates were processed for caspase activity. ( B ) SHSY-5Y neuroblastoma cells seeded on 10 cm 2 plates were treated with vehicle, carbachol (CCh; 1 mM), etoposide (25 μM) and/or atropine (0.5 μM) for 16 h, and cell lysates were processed for caspase assays. Results are means±S.E.M. ( n =3).

    Article Snippet: Etoposide, and actinomycin D were from Calbiochem (Nottingham, U.K.).

    Techniques: Activation Assay, Activity Assay

    SB203580 (SB) or SP600125 (SP) inhibit the formation of capillary-like structures and SB203580 cotreatment reduces migration and invasion of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes formed by untreated (Ctr), treated (with etoposide, LY2940042, SB203580 or SP600125 alone) and cotreated cells (etoposide plus inhibitors). The negative control is obtained by cell exposure to 10 μ M sulforaphane. Original magnification × 10. The graph reports the number of branches of the tube network formed by cells under the treatment conditions as described above. Quantitative data are the means±S.D. of three independent experiments. °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 (SB) or SP600125 (SP) inhibit the formation of capillary-like structures and SB203580 cotreatment reduces migration and invasion of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes formed by untreated (Ctr), treated (with etoposide, LY2940042, SB203580 or SP600125 alone) and cotreated cells (etoposide plus inhibitors). The negative control is obtained by cell exposure to 10 μ M sulforaphane. Original magnification × 10. The graph reports the number of branches of the tube network formed by cells under the treatment conditions as described above. Quantitative data are the means±S.D. of three independent experiments. °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Migration, Negative Control

    SB203580 (SB) reduces COX-2, ICAM-1, CXCR4 levels and MMP9 activity in etoposide-treated cells. Immunoblot analyses of COX-2 ( a ), ICAM-1 ( b ) and CXCR4 ( c ). The histograms summarize quantitative data of means±S.D. of three independent experiments. °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 (SB) reduces COX-2, ICAM-1, CXCR4 levels and MMP9 activity in etoposide-treated cells. Immunoblot analyses of COX-2 ( a ), ICAM-1 ( b ) and CXCR4 ( c ). The histograms summarize quantitative data of means±S.D. of three independent experiments. °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Activity Assay

    Etoposide activates p38MAPK, Akt and JNK. ( a ) Protein levels of PKC δ and α in cells treated with etoposide (1.25–100 μ M). Immunoblots shown are representative of three independent experiments. β -Actin is the internal loading control. ( b ) p38MAPK, JNK and Akt activation. Histograms summarize quantitative data of means±S.D. of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Etoposide activates p38MAPK, Akt and JNK. ( a ) Protein levels of PKC δ and α in cells treated with etoposide (1.25–100 μ M). Immunoblots shown are representative of three independent experiments. β -Actin is the internal loading control. ( b ) p38MAPK, JNK and Akt activation. Histograms summarize quantitative data of means±S.D. of three independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Western Blot, Activation Assay

    SB203580 cotreatment markedly reduces migration, invasion and MMP9 activity of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes in untreated (Ctr) and treated cells. Original magnification × 10. ( b ) Immunoblot analysis of VEGF. The histogram summarizes quantitative data of means±S.D. of three independent experiments °° P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: SB203580 cotreatment markedly reduces migration, invasion and MMP9 activity of etoposide-treated cells. ( a ) Formation of capillary-like structures. Representative micrographs of the complete network of tubes in untreated (Ctr) and treated cells. Original magnification × 10. ( b ) Immunoblot analysis of VEGF. The histogram summarizes quantitative data of means±S.D. of three independent experiments °° P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Migration, Activity Assay

    Effects of SB203580 (SB) cotreatment on cell viability, clonogenicity and formation of NBSs. ( a ) Left panel, cell viability. Cells were pre-treated for 1 h with the different inhibitors (0.1 μ M chelerythrine chloride (Chele), 500 nM LY2940042 (LY), 50 μ M PD98059 (PD), 10 μ M SB203580 or 4 μ M SP600125 (SP)) and then exposed to 1.25 μ M etoposide for an additional 24 h. Histogram summarizes quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Effects of SB203580 (SB) cotreatment on cell viability, clonogenicity and formation of NBSs. ( a ) Left panel, cell viability. Cells were pre-treated for 1 h with the different inhibitors (0.1 μ M chelerythrine chloride (Chele), 500 nM LY2940042 (LY), 50 μ M PD98059 (PD), 10 μ M SB203580 or 4 μ M SP600125 (SP)) and then exposed to 1.25 μ M etoposide for an additional 24 h. Histogram summarizes quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques:

    Etoposide decreases cell viability and, at high drug concentrations, inhibits the tumorigenic potential of HTLA-230 NB cells and prevents NBS formation. ( a ) Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Etoposide decreases cell viability and, at high drug concentrations, inhibits the tumorigenic potential of HTLA-230 NB cells and prevents NBS formation. ( a ) Cell viability was determined by MTT assays in cells exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: MTT Assay

    Effects of SB203580 (SB) cotreatment on viability, clonogenicity, CC133/Oct4 expression and p38MAPK activation in SK-N-SH and IMR-32 cells. ( a ) Cell viability. SK-N-SH (left panel) and IMR-32 (right panel) cells were exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

    doi: 10.1038/cddis.2013.118

    Figure Lengend Snippet: Effects of SB203580 (SB) cotreatment on viability, clonogenicity, CC133/Oct4 expression and p38MAPK activation in SK-N-SH and IMR-32 cells. ( a ) Cell viability. SK-N-SH (left panel) and IMR-32 (right panel) cells were exposed to increasing concentrations of etoposide (0.07–225 μ M) for 24 h. Histograms summarize quantitative data of means±S.D. of five independent experiments. * P

    Article Snippet: Materials Etoposide, chelerythrine chloride, LY2940042 and PD98059 were obtained from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Activation Assay

    ( A ) Western blot analysis of HO-1 protein expression after HO-1 siRNA addition to CoPP-treated macrophages. RAW 264.7 macrophages were transfected with two different HO-1 siRNA sequences (1 and 2) and nonspecific control siRNA before CoPP treatment. Lanes 1 and 2 represent HO-1 siRNA sequences 1 and 2, respectively. Note: HO-1 siRNA sequence 2 inhibited CoPP-induced HO-1 protein induction, whereas sequence 1 and nonspecific siRNA had no effect on HO-1 expression. ( B ) Western blot analysis of HO-1 and caspase-3 gene products in Ad-HO-1-transfected YPEN-1 cells. The expression of HO-1 and caspase-3 was probed with rabbit anti-mouse HO-1 ( a ) and caspase-3 ( b ) antibodies. Lane 1, YPEN-1 cells alone; lane 2, YPEN-1 cells plus etoposide (50 μ M ); lane 3, YPEN-1 cells transfected with Ad-HO-1 and HO-1 siRNA plus etoposide (50 μ M ); lane 4, YPEN-1 cells transfected with Ad-HO-1 and nonspecific siRNA plus etoposide (50 μ M ); lane 5, YPEN-1 cells transfected with Ad-HO-1; lane 6, YPEN-1 cells transfected with Ad-β-gal. Note the selectively inhibited expression of HO-1 in HO-1 siRNA-treated YPEN-1 cells (lane 3a), as compared with nonspecific siRNA and Ad-HO-1 (lanes 4a and 5a). In contrast, the expression of caspase-3 increased in cells treated with 50 μ M etoposide (lane 2b) or after HO-1 siRNA (lane 3b) or Ad-β-gal (lane 6b), as compared with nonspecific siRNA (lane 4b) or Ad-HO-1 (lane 5b). Anti-β-actin antibody was used to ensure equal protein amounts between the samples. Data shown are representative of three separate experiments.

    Journal: Human Gene Therapy

    Article Title: Small Interfering RNA Targeting Heme Oxygenase-1 (HO-1) Reinforces Liver Apoptosis Induced by Ischemia-Reperfusion Injury in Mice: HO-1 Is Necessary for Cytoprotection

    doi: 10.1089/hum.2009.049

    Figure Lengend Snippet: ( A ) Western blot analysis of HO-1 protein expression after HO-1 siRNA addition to CoPP-treated macrophages. RAW 264.7 macrophages were transfected with two different HO-1 siRNA sequences (1 and 2) and nonspecific control siRNA before CoPP treatment. Lanes 1 and 2 represent HO-1 siRNA sequences 1 and 2, respectively. Note: HO-1 siRNA sequence 2 inhibited CoPP-induced HO-1 protein induction, whereas sequence 1 and nonspecific siRNA had no effect on HO-1 expression. ( B ) Western blot analysis of HO-1 and caspase-3 gene products in Ad-HO-1-transfected YPEN-1 cells. The expression of HO-1 and caspase-3 was probed with rabbit anti-mouse HO-1 ( a ) and caspase-3 ( b ) antibodies. Lane 1, YPEN-1 cells alone; lane 2, YPEN-1 cells plus etoposide (50 μ M ); lane 3, YPEN-1 cells transfected with Ad-HO-1 and HO-1 siRNA plus etoposide (50 μ M ); lane 4, YPEN-1 cells transfected with Ad-HO-1 and nonspecific siRNA plus etoposide (50 μ M ); lane 5, YPEN-1 cells transfected with Ad-HO-1; lane 6, YPEN-1 cells transfected with Ad-β-gal. Note the selectively inhibited expression of HO-1 in HO-1 siRNA-treated YPEN-1 cells (lane 3a), as compared with nonspecific siRNA and Ad-HO-1 (lanes 4a and 5a). In contrast, the expression of caspase-3 increased in cells treated with 50 μ M etoposide (lane 2b) or after HO-1 siRNA (lane 3b) or Ad-β-gal (lane 6b), as compared with nonspecific siRNA (lane 4b) or Ad-HO-1 (lane 5b). Anti-β-actin antibody was used to ensure equal protein amounts between the samples. Data shown are representative of three separate experiments.

    Article Snippet: After washing, cells were treated with 50 μ M etoposide (Calbiochem, San Diego, CA) in YPEN-1 cultures for 2 hr or cells were treated with cobalt protoporphyrin (CoPP, an HO-1 inducer, 10 μg/ml; Porphyrin Products, Logan, UT).

    Techniques: Western Blot, Expressing, Transfection, Sequencing