etoposide eto Millipore Search Results


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  • 94
    Millipore etoposide
    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 <t>etoposide</t> treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.
    Etoposide, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8007 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore etoposide eto
    Actin polymerization increases in response to DNA damage. A. U2OS cells were treated with <t>ETO</t> (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, <t>etoposide.</t>
    Etoposide Eto, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore camptothecin cpt
    Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM <t>camptothecin</t> for 24 hr. Both floating and attached cells were
    Camptothecin Cpt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 5 fluorouracil
    The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cells HCT116 cells were treated with various commonly-used chemotherapeutic drugs, including <t>5-fluorouracil</t> (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (* P
    5 Fluorouracil, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore thapsigargin tg
    HSP47 expression affected ER-resident chaperone interactions. The expression of GRP78/Bip, CHOP/GADD135, full-length ATF6, cleaved ATF6, phospho-PERK, or IRE1 protein in NIH3T3 cells after treatment with GalNAc-bn for the indicated periods was examined by western blot analysis using anti-GRP78/Bip antibodies (A), anti-GADD153 (CHOP) antibodies (A), anti-ATF6 antibodies (B), anti-phosphoPERK antibodies (C), or anti-IRE1α antibodies (C). NIH3T3 cells treated with <t>thapsigargin</t> (Tg) were used as positive controls for these UPR-related molecule activation. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells; ATF6-fl, ATF6 full length; ATF6-c, ATF6 cleavage.
    Thapsigargin Tg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore staurosporine sts
    HSP47 expression affected mitochondria caspase-9 cleavage. (A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using an antibody against caspase-9. NIH3T3 cells treated with <t>staurosporine</t> <t>(STS)</t> were used as a positive control for caspase-9 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells. (B) CAG and BCL cells treated with STS for 12 h were examined by western blot analysis using an antibody against caspase-9. (C) Western blot analysis showed caspase-2 and GAPDH protein expression 2 d after transfection with caspase-2 siRNA. (D) CAG, BCL, and caspase-2 siRNA transfected NIH3T3 (Cas2 siRNA) cells treated with GalNAc-bn for 24 h were examined by western blot analysis using an antibody against caspase-9 after HSP47 siRNA transfection.
    Staurosporine Sts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore z vad fmk
    HSP47 expression affected mitochondria caspase-9 cleavage. (A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using an antibody against caspase-9. NIH3T3 cells treated with <t>staurosporine</t> <t>(STS)</t> were used as a positive control for caspase-9 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells. (B) CAG and BCL cells treated with STS for 12 h were examined by western blot analysis using an antibody against caspase-9. (C) Western blot analysis showed caspase-2 and GAPDH protein expression 2 d after transfection with caspase-2 siRNA. (D) CAG, BCL, and caspase-2 siRNA transfected NIH3T3 (Cas2 siRNA) cells treated with GalNAc-bn for 24 h were examined by western blot analysis using an antibody against caspase-9 after HSP47 siRNA transfection.
    Z Vad Fmk, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals etoposide
    Induction of p53 and appearance of post-mitotic G1 (2N) cells by PLK1 inhibitors occurs through the DNA damage response pathways. ( A ) HCT116-p53+/+ cells were pre-treated for 1 h with 10 μM KU55933 and/or 10 μM VE821, and subsequently treated for 24 h with 20 nM GSK461364 or, as control, 50 μM <t>etoposide.</t> Cell extracts were analyzed by western blotting. ( B ) HCT116-p53+/+ and -p53−/− cells were pre-treated for 1 h with 10 μM KU and 10μM VE821 then further treated for 24 h with 20 nM GSK461364 or 10 nM BI6727, or with DMSO as control. Cells were then harvested and analyzed by flow cytometry. ( C ) H1299 (endogenous p53-null) cells ectopically expressing wild type p53, or a S15A substitution mutant of p53, via the LacSwitch II system (Stratagene) were treated for 16 h with increasing levels of the inducer, IPTG. Cell extracts were analyzed by western blotting as indicated. ( D ) H1299-WTp53 or H1299-S15A-p53 cells were treated for 16 h with 100 μM IPTG, followed by treatment for 24 h with 20 nM GSK461364 or 10 nM BI6727, or DMSO as control. Cells were harvested and analyzed by flow cytometry. Panels A and C show cropped western blots: full length gels including molecular weight markers are provided in the Supplementary Information. The data in panels B and D are each representative of two independent experiments, each done in triplicate. Error bars represent the standard deviation of the mean.
    Etoposide, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti actin antibody
    Induction of p53 and appearance of post-mitotic G1 (2N) cells by PLK1 inhibitors occurs through the DNA damage response pathways. ( A ) HCT116-p53+/+ cells were pre-treated for 1 h with 10 μM KU55933 and/or 10 μM VE821, and subsequently treated for 24 h with 20 nM GSK461364 or, as control, 50 μM <t>etoposide.</t> Cell extracts were analyzed by western blotting. ( B ) HCT116-p53+/+ and -p53−/− cells were pre-treated for 1 h with 10 μM KU and 10μM VE821 then further treated for 24 h with 20 nM GSK461364 or 10 nM BI6727, or with DMSO as control. Cells were then harvested and analyzed by flow cytometry. ( C ) H1299 (endogenous p53-null) cells ectopically expressing wild type p53, or a S15A substitution mutant of p53, via the LacSwitch II system (Stratagene) were treated for 16 h with increasing levels of the inducer, IPTG. Cell extracts were analyzed by western blotting as indicated. ( D ) H1299-WTp53 or H1299-S15A-p53 cells were treated for 16 h with 100 μM IPTG, followed by treatment for 24 h with 20 nM GSK461364 or 10 nM BI6727, or DMSO as control. Cells were harvested and analyzed by flow cytometry. Panels A and C show cropped western blots: full length gels including molecular weight markers are provided in the Supplementary Information. The data in panels B and D are each representative of two independent experiments, each done in triplicate. Error bars represent the standard deviation of the mean.
    Anti Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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.

    Journal: Chinese Medical Journal

    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

    doi: 10.1097/CM9.0000000000000553

    Figure Lengend 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.

    Article Snippet: At approximately 90% confluence, the cells were washed with phosphate-buffered saline and then treated with 60 μg/mL etoposide (Aldrich, Sigma, St. Louis, MO 63178, USA) or with serum-free medium as a control.

    Techniques: Western Blot, Expressing

    Actin polymerization increases in response to DNA damage. A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, etoposide.

    Journal: PLoS ONE

    Article Title: Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage

    doi: 10.1371/journal.pone.0060179

    Figure Lengend Snippet: Actin polymerization increases in response to DNA damage. A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, etoposide.

    Article Snippet: Etoposide (ETO), a DNA damage inducer used in the present study, was from Sigma.

    Techniques: Software, Fluorescence, Western Blot, Immunofluorescence

    Analysis of illegitimate genomic integration of the donor plasmid in ZFN treated cells . ( A ) Effect of etoposide treatment on the levels of illegitimate integration of the PuroR expression cassette from the pDonor in the genome of 293-Flp-mEGFP cells. After transfection with pDonor, cells were either left untreated or treated with etoposide before selection with puromycin in order to quantify the levels of illegitimate integration. As a control for the selection conditions, non-transfected cells were also included. Each bar represents the mean rate of puromycin resistant (R) cells ± SD from three parallels. ( B ) Quantification of illegitimate integration in 293-Flp-mEGFP cells after transfection with pDonor along with pZFN-L and pZFN-R. Controls included non-transfected cells and cells transfected with pZFNs or pDonor individually as indicated below the bars. The rates of illegitimate integration were normalized according to variations in PE. Statistical P values (paired t-test) comparing the means of the indicated bars are shown and an asterisk marks significant difference (P

    Journal: BMC Molecular Biology

    Article Title: Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction

    doi: 10.1186/1471-2199-11-35

    Figure Lengend Snippet: Analysis of illegitimate genomic integration of the donor plasmid in ZFN treated cells . ( A ) Effect of etoposide treatment on the levels of illegitimate integration of the PuroR expression cassette from the pDonor in the genome of 293-Flp-mEGFP cells. After transfection with pDonor, cells were either left untreated or treated with etoposide before selection with puromycin in order to quantify the levels of illegitimate integration. As a control for the selection conditions, non-transfected cells were also included. Each bar represents the mean rate of puromycin resistant (R) cells ± SD from three parallels. ( B ) Quantification of illegitimate integration in 293-Flp-mEGFP cells after transfection with pDonor along with pZFN-L and pZFN-R. Controls included non-transfected cells and cells transfected with pZFNs or pDonor individually as indicated below the bars. The rates of illegitimate integration were normalized according to variations in PE. Statistical P values (paired t-test) comparing the means of the indicated bars are shown and an asterisk marks significant difference (P

    Article Snippet: Etoposide treatment of cells One day after transfection of 293-Flp-mEGFP cells with pDonor, cells were incubated overnight with growth medium containing 0.1 μM etoposide (Sigma-Aldrich).

    Techniques: Plasmid Preparation, Expressing, Transfection, Selection

    Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Expressing, Light Microscopy, Microscopy, Plasmid Preparation, Transfection, Fluorescence, Quantitative RT-PCR, shRNA, Immunofluorescence, Staining, Migration, Western Blot

    Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Transwell Invasion Assay, Wound Healing Assay

    SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Over Expression, Transfection, Expressing, Western Blot, Construct, Invasion Assay, Quantitative RT-PCR

    DBC1 did not affect SIRT1 activity in vitro in liver cancer cells. A549 and SNU-182 (A and B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM). Etoposide (20 μ M) was administered for 12 h to induce p53 hyperacetylation due to DNA damage. Knockdown of SIRT1 or DBC1 and acetylation of p53 were evaluated by western blotting with the indicated antibodies. All membranes were probed for GAPDH to confirm equal protein loading. SIRT1, silent mating type information regulation 2 homolog 1; DBC1, deleted in breast cancer-1.

    Journal: Oncology Letters

    Article Title: DBC1 does not function as a negative regulator of SIRT1 in liver cancer

    doi: 10.3892/ol.2012.875

    Figure Lengend Snippet: DBC1 did not affect SIRT1 activity in vitro in liver cancer cells. A549 and SNU-182 (A and B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM). Etoposide (20 μ M) was administered for 12 h to induce p53 hyperacetylation due to DNA damage. Knockdown of SIRT1 or DBC1 and acetylation of p53 were evaluated by western blotting with the indicated antibodies. All membranes were probed for GAPDH to confirm equal protein loading. SIRT1, silent mating type information regulation 2 homolog 1; DBC1, deleted in breast cancer-1.

    Article Snippet: To induce DNA damage, cells were treated with 20 μ M etoposide (Sigma) for 12 h.

    Techniques: Activity Assay, In Vitro, Transfection, Western Blot

    Targeted disruptions of DBC1 or SIRT1 reduced liver cancer cell viability. HepG2 (A) and SNU-182 (B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM), and then treated with 20 μ M of etoposide for 12 h. Cell viabilities were determined by MTT assay as described in Materials and methods. Data are presented as means ± standard deviation of three experiments (unpaired Student’s t-test, * P

    Journal: Oncology Letters

    Article Title: DBC1 does not function as a negative regulator of SIRT1 in liver cancer

    doi: 10.3892/ol.2012.875

    Figure Lengend Snippet: Targeted disruptions of DBC1 or SIRT1 reduced liver cancer cell viability. HepG2 (A) and SNU-182 (B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM), and then treated with 20 μ M of etoposide for 12 h. Cell viabilities were determined by MTT assay as described in Materials and methods. Data are presented as means ± standard deviation of three experiments (unpaired Student’s t-test, * P

    Article Snippet: To induce DNA damage, cells were treated with 20 μ M etoposide (Sigma) for 12 h.

    Techniques: Transfection, MTT Assay, Standard Deviation

    Trib2 overexpressing cells are chemotherapy resistant Cell viability of (A) NB4 (B) HL60 (C) U937 overexpressing Trib2 cells, in response to increasing doses of cytarabine (Ara-C), Doxocyline (Dox), daunorubicin (DNR) and Etoposide (Etop) after 24hr, determined by Trypan blue exclusion. (D) Representative apoptosis profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hr; live, DAPI−Annexin V−; early apoptotic, DAPI− Annexin V+; late apoptotic, DAPI+ Annexin V+. (E) Graph of the average fold change in cells positive for Annexin V, in U937 cells transduced with Trib2 or PHR empty vector and treated with DNR for 24hr, from 3 independent experiments. Error bars denote +/− SEM. (F) Representative mitosis FACS profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hrs. (G) Graph of fold change in mitotic index (MI), following treatment with DNR for 24hr and normalized to untreated control sample. Error bars denote +/− SEM. (H) U937 cells were transduced with control pLKO.1 or pLKO.1 shTrib2 lentivirus and treated with cytarabine (Ara-C), daunorubicin (DNR) and etoposide (Etop). Fold change in cell death (Annexin+ and/or DAPI+ cells) was determined after 24hrs. Bars represent the average of 2 independent experiments each with 3 technical replicates. Error bars denote +/− SEM.

    Journal: Oncotarget

    Article Title: Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2

    doi: 10.18632/oncotarget.24525

    Figure Lengend Snippet: Trib2 overexpressing cells are chemotherapy resistant Cell viability of (A) NB4 (B) HL60 (C) U937 overexpressing Trib2 cells, in response to increasing doses of cytarabine (Ara-C), Doxocyline (Dox), daunorubicin (DNR) and Etoposide (Etop) after 24hr, determined by Trypan blue exclusion. (D) Representative apoptosis profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hr; live, DAPI−Annexin V−; early apoptotic, DAPI− Annexin V+; late apoptotic, DAPI+ Annexin V+. (E) Graph of the average fold change in cells positive for Annexin V, in U937 cells transduced with Trib2 or PHR empty vector and treated with DNR for 24hr, from 3 independent experiments. Error bars denote +/− SEM. (F) Representative mitosis FACS profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hrs. (G) Graph of fold change in mitotic index (MI), following treatment with DNR for 24hr and normalized to untreated control sample. Error bars denote +/− SEM. (H) U937 cells were transduced with control pLKO.1 or pLKO.1 shTrib2 lentivirus and treated with cytarabine (Ara-C), daunorubicin (DNR) and etoposide (Etop). Fold change in cell death (Annexin+ and/or DAPI+ cells) was determined after 24hrs. Bars represent the average of 2 independent experiments each with 3 technical replicates. Error bars denote +/− SEM.

    Article Snippet: The cells were treated as indicated with cytarabine (Ara-C), Etoposide (Etop), doxorubicin (DOX), or daunorubicin (DNR) (Sigma-Aldrich), at the concentration indicated.

    Techniques: Acetylene Reduction Assay, Transduction, Plasmid Preparation, FACS

    miR-3196 inhibits VP-16 induced apoptosis by downregulating PUMA A. A549 cells were transfected with miR-3196 mimics (miR-3196) and/or combined with pcDNA3-PUMA (PUMA) for 24 h, followed by treatment with VP16 (100 μM) for 48 h. The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). B. H1650 cells were transfected and treated as in (A). The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). * P

    Journal: Oncotarget

    Article Title: MicroRNA-3196 is inhibited by H2AX phosphorylation and attenuates lung cancer cell apoptosis by downregulating PUMA

    doi: 10.18632/oncotarget.12794

    Figure Lengend Snippet: miR-3196 inhibits VP-16 induced apoptosis by downregulating PUMA A. A549 cells were transfected with miR-3196 mimics (miR-3196) and/or combined with pcDNA3-PUMA (PUMA) for 24 h, followed by treatment with VP16 (100 μM) for 48 h. The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). B. H1650 cells were transfected and treated as in (A). The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). * P

    Article Snippet: Cells were grown to 80% confluence and then serum-deprived for 12 h prior to etoposide (VP16) (Sigma–Aldrich, St Louis, MO, USA) stimulation.

    Techniques: Transfection

    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

    Quantification of apoptosis in PAX3 siRNA transfected SH-SY5Y (A and B) and SH-EP1 (C and D) cells following the treatment with chemotherapeutic drugs. (A) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-SY5Y cells which were exposed to drugs at IC 50 for 24 hrs for annexin V (green) and propidium iodide (red). (B) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-SY5Y cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 80 /IC 90 for 6, 24, 48 and 72 hrs. (C) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-EP1 cells which were exposed to drugs at IC 90 for 6 hrs for annexin V (green) and propidium iodide (red). (D) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-EP1 cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 90 for 6, 24, 48 and 72 hrs. Scale bars represent 20 μm. Error bars represent mean ± SD for two independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: PAX3 in neuroblastoma: oncogenic potential, chemosensitivity and signalling pathways

    doi: 10.1111/jcmm.12155

    Figure Lengend Snippet: Quantification of apoptosis in PAX3 siRNA transfected SH-SY5Y (A and B) and SH-EP1 (C and D) cells following the treatment with chemotherapeutic drugs. (A) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-SY5Y cells which were exposed to drugs at IC 50 for 24 hrs for annexin V (green) and propidium iodide (red). (B) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-SY5Y cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 80 /IC 90 for 6, 24, 48 and 72 hrs. (C) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-EP1 cells which were exposed to drugs at IC 90 for 6 hrs for annexin V (green) and propidium iodide (red). (D) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-EP1 cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 90 for 6, 24, 48 and 72 hrs. Scale bars represent 20 μm. Error bars represent mean ± SD for two independent experiments. * P

    Article Snippet: Cytotoxicity induced by chemotherapeutic drugs Etoposide and cisplatin were dissolved in dimethyl sulphoxide (DMSO) and vincristine in methanol (all from Sigma-Aldrich), stored at −20°C, further diluted in growth medium immediately before use (the final concentrations of DMSO and methanol were < 0.04).

    Techniques: Transfection, Immunofluorescence, Staining

    Effects of controls CTP (camptothecin topo I inhibitor), ETP (etoposide topo II poison), and SBZ (Sobuzoxane topo II inhibitor), and extracts from A. cuspidata, A. integrifolium , G. mexicanum , and L. Achyranthifolia on JN394t-1 (30°C), JN395t2-5 (25°C), JN394t2-4 (25°C), and JN394t2-4 (30°C) growth. G. mexicanum was identified as topoisomerase inhibitor I, meanwhile these extracts showed similar behavior to that of SBZ, a topo II inhibitor (catalytic). JN395t2-5 (25°C), JN394t2-4 (25°C), and JN394t2-4 (30°C) assays with G. mexicanum were not determinate (ND).

    Journal: African Journal of Traditional, Complementary, and Alternative Medicines

    Article Title: EVALUATION OF ANTICANCER POTENTIAL OF EIGHT VEGETAL SPECIES FROM THE STATE OF OAXACA

    doi: 10.21010/ajtcam.v14i3.7

    Figure Lengend Snippet: Effects of controls CTP (camptothecin topo I inhibitor), ETP (etoposide topo II poison), and SBZ (Sobuzoxane topo II inhibitor), and extracts from A. cuspidata, A. integrifolium , G. mexicanum , and L. Achyranthifolia on JN394t-1 (30°C), JN395t2-5 (25°C), JN394t2-4 (25°C), and JN394t2-4 (30°C) growth. G. mexicanum was identified as topoisomerase inhibitor I, meanwhile these extracts showed similar behavior to that of SBZ, a topo II inhibitor (catalytic). JN395t2-5 (25°C), JN394t2-4 (25°C), and JN394t2-4 (30°C) assays with G. mexicanum were not determinate (ND).

    Article Snippet: Methanol (HPLC grade), methanol (spectrophotometric grade), camptothecin (CPT), etoposide (ETP), dimethyl sulfoxide (DMSO-Hybri-Max), adenine hemisulfate salt, chloranphenicol, nystatin, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), benzo [a] pyrene (B[a]P), and sodium azide (NaN3) were obtained from Sigma Chemical (St. Louis, MO).

    Techniques:

    Effects of controls CPT (camptothecin topo I inhibitor), and ETP (etoposide topo II poison), and extracts on JN394 (30°C) and JN362a (30°C) growth. Treatments with similar behaviors to CPT or ETP are A. cuspidata , A integrifolium , G. mexicanum , and L. achyranthifolia , which inhibited growth of JN394 and promoted or did not affect that of JN362a. A. aurantium , A. aurantium R , T. densiflora and T. densiflora R were toxic to both strains.

    Journal: African Journal of Traditional, Complementary, and Alternative Medicines

    Article Title: EVALUATION OF ANTICANCER POTENTIAL OF EIGHT VEGETAL SPECIES FROM THE STATE OF OAXACA

    doi: 10.21010/ajtcam.v14i3.7

    Figure Lengend Snippet: Effects of controls CPT (camptothecin topo I inhibitor), and ETP (etoposide topo II poison), and extracts on JN394 (30°C) and JN362a (30°C) growth. Treatments with similar behaviors to CPT or ETP are A. cuspidata , A integrifolium , G. mexicanum , and L. achyranthifolia , which inhibited growth of JN394 and promoted or did not affect that of JN362a. A. aurantium , A. aurantium R , T. densiflora and T. densiflora R were toxic to both strains.

    Article Snippet: Methanol (HPLC grade), methanol (spectrophotometric grade), camptothecin (CPT), etoposide (ETP), dimethyl sulfoxide (DMSO-Hybri-Max), adenine hemisulfate salt, chloranphenicol, nystatin, ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), benzo [a] pyrene (B[a]P), and sodium azide (NaN3) were obtained from Sigma Chemical (St. Louis, MO).

    Techniques: Cycling Probe Technology

    Representative MS/MS spectra of Ε p53. Peptides prepared from p53 isolated from HFFs treated with etoposide were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.”

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53 *

    doi: 10.1074/mcp.M113.030254

    Figure Lengend Snippet: Representative MS/MS spectra of Ε p53. Peptides prepared from p53 isolated from HFFs treated with etoposide were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.”

    Article Snippet: For the comparison of p53 protein concentrations in infected and etoposide-treated cells, etoposide (Sigma) was diluted to 50 m m in dimethyl sulfoxide and added to medium at a final concentration of 125 μ m .

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    Analysis of DNA binding by p53. The relative concentrations of p53 present in whole cell extracts of HFFs infected with 100 pfu/cell AdEasyE1(Δ)2347 (ΔE1B p53) or exposed to 125 μ m etoposide (E p53) for 44 h were determined via

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53 *

    doi: 10.1074/mcp.M113.030254

    Figure Lengend Snippet: Analysis of DNA binding by p53. The relative concentrations of p53 present in whole cell extracts of HFFs infected with 100 pfu/cell AdEasyE1(Δ)2347 (ΔE1B p53) or exposed to 125 μ m etoposide (E p53) for 44 h were determined via

    Article Snippet: For the comparison of p53 protein concentrations in infected and etoposide-treated cells, etoposide (Sigma) was diluted to 50 m m in dimethyl sulfoxide and added to medium at a final concentration of 125 μ m .

    Techniques: Binding Assay, Infection

    Etoposide induced p53 activity is dampened in chronic hypoxia. D283-MED cells were incubated in 1% O 2 or 21% O 2 for 1 day or 5 days, prior to etoposide treatment where indicated. Three p53 target genes, MDM2, PUMA and p21 were assessed by qPCR. ( a ) Basal levels of p53 target genes in hypoxia without etoposide treatment were measured. The mRNA levels were normalised with the house-keeping gene cyclophilin A. ( b ) Levels of MDM2, p21 and PUMA mRNA with or without etoposide treatment. Data represented as normalised to housekeeping gene (cyclophilin A) and fold change with respect to the untreated control. Data are representative of three independent experiments, error bars are SD of a single experiment. ( c ) Total p53 and phosphorylated p53 serine 15 levels assessed by western blot. ( d ) Densitometry quantification of the band intensity was analysed by ImageJ from 3 independent experiments. Plot represents p53 serine 15 over the p53 total

    Journal: BMC Cancer

    Article Title: Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells

    doi: 10.1186/s12885-019-5476-9

    Figure Lengend Snippet: Etoposide induced p53 activity is dampened in chronic hypoxia. D283-MED cells were incubated in 1% O 2 or 21% O 2 for 1 day or 5 days, prior to etoposide treatment where indicated. Three p53 target genes, MDM2, PUMA and p21 were assessed by qPCR. ( a ) Basal levels of p53 target genes in hypoxia without etoposide treatment were measured. The mRNA levels were normalised with the house-keeping gene cyclophilin A. ( b ) Levels of MDM2, p21 and PUMA mRNA with or without etoposide treatment. Data represented as normalised to housekeeping gene (cyclophilin A) and fold change with respect to the untreated control. Data are representative of three independent experiments, error bars are SD of a single experiment. ( c ) Total p53 and phosphorylated p53 serine 15 levels assessed by western blot. ( d ) Densitometry quantification of the band intensity was analysed by ImageJ from 3 independent experiments. Plot represents p53 serine 15 over the p53 total

    Article Snippet: Reagents Etoposide (E1383) was from Sigma.

    Techniques: Activity Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot

    Chronic hypoxia reduces ATM activation after etoposide treatment. D283-MED cells were pre-incubated in 21% O 2 , 1% O 2 or 0.1% O 2 prior to treatment with 20 μM etoposide for 4 h, or cells were left untreated as a control. Levels of ATM and ATM serine 1981 phosphorylation were determined using 3 independent western blots. The plots represent the densitometry of a representative western blot ( a ) measured using Image J

    Journal: BMC Cancer

    Article Title: Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells

    doi: 10.1186/s12885-019-5476-9

    Figure Lengend Snippet: Chronic hypoxia reduces ATM activation after etoposide treatment. D283-MED cells were pre-incubated in 21% O 2 , 1% O 2 or 0.1% O 2 prior to treatment with 20 μM etoposide for 4 h, or cells were left untreated as a control. Levels of ATM and ATM serine 1981 phosphorylation were determined using 3 independent western blots. The plots represent the densitometry of a representative western blot ( a ) measured using Image J

    Article Snippet: Reagents Etoposide (E1383) was from Sigma.

    Techniques: Activation Assay, Incubation, Western Blot

    Etoposide efficacy is not affected by hypoxia. D283-MED cells were incubated in 1% O 2 for 5 days or in 21% O 2 prior to etoposide (20 μM) treatment for indicated time points. ( a ) ɣH2AX bound secondary Cy3 antibodies were detected by immunofluorescence, staining intensity was quantified for individual cells using AQM analysis. Data shown are the mean ± S.E.M of three independent experiments ( n > 350 cells). One-way ANOVA followed by Bonferroni test was performed (*indicates p

    Journal: BMC Cancer

    Article Title: Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells

    doi: 10.1186/s12885-019-5476-9

    Figure Lengend Snippet: Etoposide efficacy is not affected by hypoxia. D283-MED cells were incubated in 1% O 2 for 5 days or in 21% O 2 prior to etoposide (20 μM) treatment for indicated time points. ( a ) ɣH2AX bound secondary Cy3 antibodies were detected by immunofluorescence, staining intensity was quantified for individual cells using AQM analysis. Data shown are the mean ± S.E.M of three independent experiments ( n > 350 cells). One-way ANOVA followed by Bonferroni test was performed (*indicates p

    Article Snippet: Reagents Etoposide (E1383) was from Sigma.

    Techniques: Incubation, Immunofluorescence, Staining

    Chronic hypoxia-induces treatment resistance in MB and glioblastoma cells. Cell viability was determined using an MTS assay, with absorbance normalised to untreated control. Two MB and one glioblastoma cell lines were pre-cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) for the indicated time points prior to etoposide, cisplatin or X-ray irradiation treatment. Cells were maintained in 1% O 2 during etoposide [20–100 μM] and cisplatin [1–50 μg/ml] treatment. ( a ) D283-MED cells treated with etoposide ( b ) MEB-Med8A treated with etoposide ( c ) U87MG cells treated with etoposide. ( d ) D283-MED treated with cisplatin (1–5 μg/ml). ( e ) MEB-Med8A treated with cisplatin (1–5 μg/ml) ( f ) U87MG treated with cisplatin (30–50 μg/ml) ( g ) X-ray irradiation treatment was conducted in 21% O 2 with doses of 30 Gy (D283-MED), 50 Gy (MEB-Med8A) and 2 × 80 Gy dose (U87MG), with 48 h incubation post-treatment. The different doses is to account for different cell sensitivity to irradiation. Data are shown as the mean ± S.E.M of at least 3 independent experiments. A student t-test was performed where (*) indicates statistical significance with p

    Journal: BMC Cancer

    Article Title: Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells

    doi: 10.1186/s12885-019-5476-9

    Figure Lengend Snippet: Chronic hypoxia-induces treatment resistance in MB and glioblastoma cells. Cell viability was determined using an MTS assay, with absorbance normalised to untreated control. Two MB and one glioblastoma cell lines were pre-cultured in normoxia (21% O 2 ) or hypoxia (1% O 2 ) for the indicated time points prior to etoposide, cisplatin or X-ray irradiation treatment. Cells were maintained in 1% O 2 during etoposide [20–100 μM] and cisplatin [1–50 μg/ml] treatment. ( a ) D283-MED cells treated with etoposide ( b ) MEB-Med8A treated with etoposide ( c ) U87MG cells treated with etoposide. ( d ) D283-MED treated with cisplatin (1–5 μg/ml). ( e ) MEB-Med8A treated with cisplatin (1–5 μg/ml) ( f ) U87MG treated with cisplatin (30–50 μg/ml) ( g ) X-ray irradiation treatment was conducted in 21% O 2 with doses of 30 Gy (D283-MED), 50 Gy (MEB-Med8A) and 2 × 80 Gy dose (U87MG), with 48 h incubation post-treatment. The different doses is to account for different cell sensitivity to irradiation. Data are shown as the mean ± S.E.M of at least 3 independent experiments. A student t-test was performed where (*) indicates statistical significance with p

    Article Snippet: Reagents Etoposide (E1383) was from Sigma.

    Techniques: MTS Assay, Cell Culture, Irradiation, Incubation

    Rit is activated in response to diverse extracellular stimuli. (A) Stress-mediated Rit activation. PC6 cells were transfected with Flag–hRit-WT (0.5 μg) and starved for 5 h prior to stimulation with ActD (2 μM), etoposide (40 μM),

    Journal: Molecular and Cellular Biology

    Article Title: A Rit GTPase-p38 Mitogen-Activated Protein Kinase Survival Pathway Confers Resistance to Cellular Stress ▿

    doi: 10.1128/MCB.01380-10

    Figure Lengend Snippet: Rit is activated in response to diverse extracellular stimuli. (A) Stress-mediated Rit activation. PC6 cells were transfected with Flag–hRit-WT (0.5 μg) and starved for 5 h prior to stimulation with ActD (2 μM), etoposide (40 μM),

    Article Snippet: The following were purchased: nerve growth factor (NGF), epidermal growth factor (EGF), bone morphogenetic protein 7 (BMP7), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) (R & D systems); PACAP38 (Bachem), actinomycin D (ActD), etoposide (ET), and staurosporine (STS) (CalBiochem); hydrogen peroxide (H2 O2 ) (Sigma); kinase-specific inhibitors SB203580 (Tocris), PD98059, and LY294002 and AKT inhibitor V (triciribine) (CalBiochem); antibodies against caspase-3, phospho-p38, p38, phospho-AKT (Ser473 and Thr308 ), AKT, phospho-MK2 (Thr334 ), MK2, phospho-HSP27 (Ser82 ), and HSP27 (Cell Signaling); antibody against phospho-JNK1/2 (pTpY183/185 ) (Invitrogen); and antibodies against actin and Flag (Sigma).

    Techniques: Activation Assay, Transfection

    OTIs designed against a patient-observed PML-RARA translocation increase DNA cleavage mediated by human type II topoisomerases. ( A ) Sequences of the top and bottom strands of each PML-RARA duplex are shown. The blue portion corresponds to the segment derived from the PML gene, and the orange portion corresponds to the segment derived from the RARA gene. The yellow box indicates the position of the tethered etoposide core on each OTI (bottom strand). The OTIs were 50, 30 or 20 bases in length (black lines below the diagram). Arrows indicate sites of DNA cleavage induced by free etoposide (blue) and the translocation OTIs (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML-RARA top/target strand hybridized to an unmodified PML-RARA bottom strand in the presence of free etoposide or of the radiolabeled PML-RARA top strand hybridized to a 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand. Lanes 1–5 contain the unmodified PML-RARA duplex in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML-RARA top strand hybridized with the 50-mer OTI. Lanes 10 and 11 contain the unmodified top strand hybridized with the 30-mer OTI. Lanes 13 and 14 contain the unmodified top strand hybridized with the 20-mer OTI. Lanes 6, 10, 13, and 15 contain reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of enzyme-mediated DNA cleavage. DNA cleavage at each site is normalized to the cleavage observed at site 24–25 in reactions containing an unmodified duplex in the absence of etoposide (lane 2). Results with unmodified PML – RARA duplex in the presence of 500 μM free etoposide (blue) or with unmodified top strand hybridized with 50-mer OTI (yellow), 30-mer OTI (orange), or 20-mer OTI (green) bottom strand are shown. Error bars represent the standard deviation of at least three independent experiments. Significance was determined by paired t -tests. P -values are indicated by asterisks (* P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTIs designed against a patient-observed PML-RARA translocation increase DNA cleavage mediated by human type II topoisomerases. ( A ) Sequences of the top and bottom strands of each PML-RARA duplex are shown. The blue portion corresponds to the segment derived from the PML gene, and the orange portion corresponds to the segment derived from the RARA gene. The yellow box indicates the position of the tethered etoposide core on each OTI (bottom strand). The OTIs were 50, 30 or 20 bases in length (black lines below the diagram). Arrows indicate sites of DNA cleavage induced by free etoposide (blue) and the translocation OTIs (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML-RARA top/target strand hybridized to an unmodified PML-RARA bottom strand in the presence of free etoposide or of the radiolabeled PML-RARA top strand hybridized to a 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand. Lanes 1–5 contain the unmodified PML-RARA duplex in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML-RARA top strand hybridized with the 50-mer OTI. Lanes 10 and 11 contain the unmodified top strand hybridized with the 30-mer OTI. Lanes 13 and 14 contain the unmodified top strand hybridized with the 20-mer OTI. Lanes 6, 10, 13, and 15 contain reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of enzyme-mediated DNA cleavage. DNA cleavage at each site is normalized to the cleavage observed at site 24–25 in reactions containing an unmodified duplex in the absence of etoposide (lane 2). Results with unmodified PML – RARA duplex in the presence of 500 μM free etoposide (blue) or with unmodified top strand hybridized with 50-mer OTI (yellow), 30-mer OTI (orange), or 20-mer OTI (green) bottom strand are shown. Error bars represent the standard deviation of at least three independent experiments. Significance was determined by paired t -tests. P -values are indicated by asterisks (* P

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Translocation Assay, Derivative Assay, Standard Deviation

    Structure-guided design of an OTI. ( A ) Schematic illustrating domains of type II topoisomerases used to determine the crystal structure of human topoisomerase IIβ covalently attached to DNA (green) in the presence of etoposide (orange). Domains pictured are TOPRIM (Top), winged helix domain (WHD), tower domain (Tow), and exit gate domain (Ex). ( B ) Schematic of topoisomerase II function. Protein protomer subunits are shown in blue and gray. T DNA, transport double helix (black); G DNA, gate double helix (green). ( C, D ) Detail from the crystal structure of a topoisomerase IIβ cleavage complex with two bound etoposide molecules (orange) stabilizing a double-stranded DNA (green) break; PDB code 3QX3. For clarity, in panel C, only the Cα trace of the protein subunits (blue and black lines) and catalytic tyrosines (blue and gray sticks) are shown. In panel D, only the catalytic tyrosine residues that cleave the DNA are shown. The conventional numbering scheme used for DNA cleavage complexes formed by type II topoisomerases is shown. The enzyme cleaves between the -1 and the +1 on each strand. The numbering on the two strands in the double helix is differentiated by the presence or absence of asterisks. The catalytic tyrosine residues are covalently attached to the DNA at the +1 positions. ( E , F ) Model of a cleavage complex with one bound etoposide molecule stabilizing a single-stranded DNA break. The cleaved DNA strand is indicated by asterisks. The protein subunits shown are the same as those in C and D. ( G ) Chemical (left) and modeled (right) structure of the etoposide core (DEPT) linked to the pyrimidine base. ( H ) OTI28 (orange strand) modeled with the modified cytosine base in the +5* position, stabilizing DNA scission at the 23–24 site (–1 to +1) on the cleaved target strand (green). Structural figures were drawn with Pymol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: Structure-guided design of an OTI. ( A ) Schematic illustrating domains of type II topoisomerases used to determine the crystal structure of human topoisomerase IIβ covalently attached to DNA (green) in the presence of etoposide (orange). Domains pictured are TOPRIM (Top), winged helix domain (WHD), tower domain (Tow), and exit gate domain (Ex). ( B ) Schematic of topoisomerase II function. Protein protomer subunits are shown in blue and gray. T DNA, transport double helix (black); G DNA, gate double helix (green). ( C, D ) Detail from the crystal structure of a topoisomerase IIβ cleavage complex with two bound etoposide molecules (orange) stabilizing a double-stranded DNA (green) break; PDB code 3QX3. For clarity, in panel C, only the Cα trace of the protein subunits (blue and black lines) and catalytic tyrosines (blue and gray sticks) are shown. In panel D, only the catalytic tyrosine residues that cleave the DNA are shown. The conventional numbering scheme used for DNA cleavage complexes formed by type II topoisomerases is shown. The enzyme cleaves between the -1 and the +1 on each strand. The numbering on the two strands in the double helix is differentiated by the presence or absence of asterisks. The catalytic tyrosine residues are covalently attached to the DNA at the +1 positions. ( E , F ) Model of a cleavage complex with one bound etoposide molecule stabilizing a single-stranded DNA break. The cleaved DNA strand is indicated by asterisks. The protein subunits shown are the same as those in C and D. ( G ) Chemical (left) and modeled (right) structure of the etoposide core (DEPT) linked to the pyrimidine base. ( H ) OTI28 (orange strand) modeled with the modified cytosine base in the +5* position, stabilizing DNA scission at the 23–24 site (–1 to +1) on the cleaved target strand (green). Structural figures were drawn with Pymol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Modification

    ). ( A ) Cleavage between bases 24–25 is depicted on the target (top) strand (green). ( B ) Cleavage between bases 23–24 is depicted on the target strand (green). The bottom (OTI) strand is shown in orange. The tethered etoposide core is shown in yellow (carbons, yellow; nitrogen, blue; oxygen, red). A Cα trace is shown for the two topoisomerase II subunits (blue and black lines) in the top panels. The bottom panels include a semi-transparent molecular surface, illustrating that the linker does not clash with the protein. The sequence diagram (middle) shows the position of the tethered etoposide core on OTI28 (yellow box). Black arrows indicate the cleavage sites.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: ). ( A ) Cleavage between bases 24–25 is depicted on the target (top) strand (green). ( B ) Cleavage between bases 23–24 is depicted on the target strand (green). The bottom (OTI) strand is shown in orange. The tethered etoposide core is shown in yellow (carbons, yellow; nitrogen, blue; oxygen, red). A Cα trace is shown for the two topoisomerase II subunits (blue and black lines) in the top panels. The bottom panels include a semi-transparent molecular surface, illustrating that the linker does not clash with the protein. The sequence diagram (middle) shows the position of the tethered etoposide core on OTI28 (yellow box). Black arrows indicate the cleavage sites.

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Sequencing

    OTI28 inhibits DNA ligation and stabilizes cleavage complexes similarly to free etoposide. ( A ) Enzyme-mediated ligation of DNA. ( B ) Persistence of cleavage complexes. For both A and B, cleavage results of the unmodified PML duplex in the presence of 500 μM free etoposide are shown in blue and those with an unmodified PML top/target strand hybridized to OTI28 are shown in yellow. Error bars represent the standard deviation of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTI28 inhibits DNA ligation and stabilizes cleavage complexes similarly to free etoposide. ( A ) Enzyme-mediated ligation of DNA. ( B ) Persistence of cleavage complexes. For both A and B, cleavage results of the unmodified PML duplex in the presence of 500 μM free etoposide are shown in blue and those with an unmodified PML top/target strand hybridized to OTI28 are shown in yellow. Error bars represent the standard deviation of at least three independent experiments.

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: DNA Ligation, Ligation, Standard Deviation

    An oligonucleotide-linked etoposide core increases topoisomerase II-mediated DNA cleavage. ( A ) The central 30 base pairs of a double stranded 50-mer oligonucleotide sequence corresponding to bases 1471–1500 (top strand) of PML intron 6 is shown. The yellow box denotes the position of the tethered etoposide core and linker moieties on OTI28 or LIN28. Arrows indicate sites of DNA cleavage induced by free etoposide (blue) or OTI28 (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of free etoposide or hybridized to OTI28 (bottom strand). For each gel, lane 1 contains the unmodified PML oligonucleotide. Lanes 2–5 contain the unmodified PML duplex treated with 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML top strand hybridized with OTI28. Lanes 10 and 11 contain the unmodified top strand duplexed with LIN28 (bottom strand oligonucleotide that contains the linker at position 28 with no attached etoposide core). Lanes 6, 9, and 12 contain reference (R) oligonucleotides that were 24, 23 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of DNA cleavage mediated by topoisomerase IIα (left) and topoisomerase IIβ (right). DNA cleavage at each site was normalized to the cleavage observed at site 24–25 in reactions containing unmodified duplex in the absence of etoposide (lane 2). Cleavage results of the unmodified duplex in the presence of 500 μM free etoposide are shown in blue (lane 5) and those with an unmodified top strand hybridized to OTI28 are shown in yellow (lane 8). Error bars represent the standard error of the mean of an average of two to five independent experiments. Significance was determined by paired t-tests. P -values are indicated by asterisks (* P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: An oligonucleotide-linked etoposide core increases topoisomerase II-mediated DNA cleavage. ( A ) The central 30 base pairs of a double stranded 50-mer oligonucleotide sequence corresponding to bases 1471–1500 (top strand) of PML intron 6 is shown. The yellow box denotes the position of the tethered etoposide core and linker moieties on OTI28 or LIN28. Arrows indicate sites of DNA cleavage induced by free etoposide (blue) or OTI28 (yellow). ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of free etoposide or hybridized to OTI28 (bottom strand). For each gel, lane 1 contains the unmodified PML oligonucleotide. Lanes 2–5 contain the unmodified PML duplex treated with 0–500 μM free etoposide. Lanes 7 and 8 contain the unmodified PML top strand hybridized with OTI28. Lanes 10 and 11 contain the unmodified top strand duplexed with LIN28 (bottom strand oligonucleotide that contains the linker at position 28 with no attached etoposide core). Lanes 6, 9, and 12 contain reference (R) oligonucleotides that were 24, 23 and 19 bases in length. Gels are representative of at least three independent experiments. ( C ) Quantification of the relative levels of DNA cleavage mediated by topoisomerase IIα (left) and topoisomerase IIβ (right). DNA cleavage at each site was normalized to the cleavage observed at site 24–25 in reactions containing unmodified duplex in the absence of etoposide (lane 2). Cleavage results of the unmodified duplex in the presence of 500 μM free etoposide are shown in blue (lane 5) and those with an unmodified top strand hybridized to OTI28 are shown in yellow (lane 8). Error bars represent the standard error of the mean of an average of two to five independent experiments. Significance was determined by paired t-tests. P -values are indicated by asterisks (* P

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Sequencing

    OTI28 induces lower levels of DNA cleavage mediated by an etoposide-resistant mutant yeast topoisomerase II (H1011Y) as compared to wild-type yeast topoisomerase II. Quantification of the relative levels of enzyme-mediated DNA cleavage at site 24–25 (indicated as the band labeled 24 in the inset) mediated by wild-type (yTop2WT) and H1011Y mutant (yTop2H1011Y) yeast topoisomerase II on an unmodified PML top strand hybridized to OTI28 (graph: +enz, red; inset: +WT, +H1011Y). DNA cleavage is normalized to background levels of DNA when no enzyme is present (graph: -enz, blue; inset: -WT, -H1011Y). Error bars represent the standard deviation of three independent experiments. Significance was determined by a paired t -test. P -values are indicated by asterisks (*** P

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTI28 induces lower levels of DNA cleavage mediated by an etoposide-resistant mutant yeast topoisomerase II (H1011Y) as compared to wild-type yeast topoisomerase II. Quantification of the relative levels of enzyme-mediated DNA cleavage at site 24–25 (indicated as the band labeled 24 in the inset) mediated by wild-type (yTop2WT) and H1011Y mutant (yTop2H1011Y) yeast topoisomerase II on an unmodified PML top strand hybridized to OTI28 (graph: +enz, red; inset: +WT, +H1011Y). DNA cleavage is normalized to background levels of DNA when no enzyme is present (graph: -enz, blue; inset: -WT, -H1011Y). Error bars represent the standard deviation of three independent experiments. Significance was determined by a paired t -test. P -values are indicated by asterisks (*** P

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Mutagenesis, Labeling, Standard Deviation

    Moving the position of the linked etoposide core along the bottom strand (OTI) sequence alters the topoisomerase II-mediated cleavage pattern of the top strand. ( A ) Sequences of the top/target and bottom PML strands are shown. The different-colored boxes indicate the position of the tethered etoposide core in each OTI (bottom strand), including OTI28 (yellow), OTI29 (orange), OTI33 (green), and OTI23 (purple). Arrows indicate the cleavage sites induced by each OTI (shown by corresponding colors). Large arrows indicate the major site of cleavage. T and B (first column) indicate top and bottom strands, respectively. ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of 0–500 μM free etoposide (lanes 2–3) or hybridized to the bottom strands OTI28 (lanes 5–6), OTI29 (lanes 8–9), OTI33 (lanes 11–12), or OTI23 (lanes 14–15). For each gel, lane 1 contains an unmodified PML duplex. Reference (R) oligonucleotides are 24, 23, 20 and 19 bases long. Gels are representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: Moving the position of the linked etoposide core along the bottom strand (OTI) sequence alters the topoisomerase II-mediated cleavage pattern of the top strand. ( A ) Sequences of the top/target and bottom PML strands are shown. The different-colored boxes indicate the position of the tethered etoposide core in each OTI (bottom strand), including OTI28 (yellow), OTI29 (orange), OTI33 (green), and OTI23 (purple). Arrows indicate the cleavage sites induced by each OTI (shown by corresponding colors). Large arrows indicate the major site of cleavage. T and B (first column) indicate top and bottom strands, respectively. ( B ) Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα, left) and topoisomerase IIβ (hTIIβ, right) of the radiolabeled, unmodified PML top strand hybridized to an unmodified PML bottom strand in the presence of 0–500 μM free etoposide (lanes 2–3) or hybridized to the bottom strands OTI28 (lanes 5–6), OTI29 (lanes 8–9), OTI33 (lanes 11–12), or OTI23 (lanes 14–15). For each gel, lane 1 contains an unmodified PML duplex. Reference (R) oligonucleotides are 24, 23, 20 and 19 bases long. Gels are representative of at least three independent experiments.

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Sequencing

    An oligonucleotide with an abasic site analog at position 28 generates a different DNA cleavage pattern than does OTI28. Lanes 1–3 contain a radiolabeled unmodified PML top/target strand hybridized to an unmodified PML bottom strand in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 4 and 5 contain a radiolabeled PML top strand hybridized with OTI28 (bottom strand). Lanes 6 and 7 contain a radiolabeled unmodified PML top strand hybridized to a bottom strand oligonucleotide containing an abasic site analog at position 28 (AP28). Lane 8 contains reference (R) oligonucleotides that are 24, 23 and 19 bases in length. The gel is representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: An oligonucleotide with an abasic site analog at position 28 generates a different DNA cleavage pattern than does OTI28. Lanes 1–3 contain a radiolabeled unmodified PML top/target strand hybridized to an unmodified PML bottom strand in the absence of enzyme, or in the presence of enzyme and 0–500 μM free etoposide. Lanes 4 and 5 contain a radiolabeled PML top strand hybridized with OTI28 (bottom strand). Lanes 6 and 7 contain a radiolabeled unmodified PML top strand hybridized to a bottom strand oligonucleotide containing an abasic site analog at position 28 (AP28). Lane 8 contains reference (R) oligonucleotides that are 24, 23 and 19 bases in length. The gel is representative of at least three independent experiments.

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques:

    OTIs that incorporate an APL patient-derived PML-RARA translocation sequence do not increase DNA cleavage mediated by human type II topoisomerases when they are hybridized with the parental PML or RARA sequences. Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα) ( A ) and topoisomerase IIβ (hTIIβ) ( B ) of the radiolabeled top strand of an unmodified PML-RARA duplex in the presence of free etoposide, a radiolabeled, unmodified PML or RARA top strand hybridized to a PML-RARA bottom strand in the presence of free etoposide, or a radiolabeled, unmodified PML or RARA top strand hybridized to a 50-mer OTI, a 30-mer OTI, or a 20-mer PML-RARA OTI bottom strand. For each gel, lanes 1–3 contain unmodified PML-RARA top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 4–6 contain unmodified parental PML top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 7–12 contain the unmodified parental PML top strand hybridized to the 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 14–16 contain unmodified parental RARA top strand hybridized to the 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 17–22 contain the unmodified parental RARA top strand hybridized to the 50-mer, 30-mer, or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 13 and 23 contain a combination of reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences

    doi: 10.1093/nar/gky072

    Figure Lengend Snippet: OTIs that incorporate an APL patient-derived PML-RARA translocation sequence do not increase DNA cleavage mediated by human type II topoisomerases when they are hybridized with the parental PML or RARA sequences. Comparison of DNA cleavage mediated by human topoisomerase IIα (hTIIα) ( A ) and topoisomerase IIβ (hTIIβ) ( B ) of the radiolabeled top strand of an unmodified PML-RARA duplex in the presence of free etoposide, a radiolabeled, unmodified PML or RARA top strand hybridized to a PML-RARA bottom strand in the presence of free etoposide, or a radiolabeled, unmodified PML or RARA top strand hybridized to a 50-mer OTI, a 30-mer OTI, or a 20-mer PML-RARA OTI bottom strand. For each gel, lanes 1–3 contain unmodified PML-RARA top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 4–6 contain unmodified parental PML top/target strand hybridized to the unmodified 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 7–12 contain the unmodified parental PML top strand hybridized to the 50-mer, 30-mer or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 14–16 contain unmodified parental RARA top strand hybridized to the 50-mer PML-RARA bottom strand, in the absence of enzyme, or in the presence of enzyme and 0–500 μM etoposide. Lanes 17–22 contain the unmodified parental RARA top strand hybridized to the 50-mer, 30-mer, or 20-mer PML-RARA OTI bottom strand in the absence or presence of enzyme. Lanes 13 and 23 contain a combination of reference (R) oligonucleotides 24, 23, 20 and 19 bases in length. Gels are representative of at least three independent experiments.

    Article Snippet: Analytical grade etoposide was purchased from Sigma-Aldrich and stored at room temperature as a 40 mM stock solution in 100% DMSO.

    Techniques: Derivative Assay, Translocation Assay, Sequencing

    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)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques:

    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)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques: 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)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques: 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

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

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

    Etoposide deregulates cluster of genomic and miRNA biomarkers in hiPSC-CMs. Heat maps showing ETP-induced deregulation of genomic and miRNA markers in CMs. a Represents genes downregulated after ETP treatment ( n = 3, fold change ≤ − 1.8). b Represents genes upregulated after ETP treatment ( n = 3, fold change ≥ 1.8). c Represents genes with no significant change in expression level after ETP treatment ( n = 3). d Venn diagram showing commonly deregulated genes in groups with different ETP concentrations ( n = 3). e Represents deregulated miRNA markers ( n = 3)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend Snippet: Etoposide deregulates cluster of genomic and miRNA biomarkers in hiPSC-CMs. Heat maps showing ETP-induced deregulation of genomic and miRNA markers in CMs. a Represents genes downregulated after ETP treatment ( n = 3, fold change ≤ − 1.8). b Represents genes upregulated after ETP treatment ( n = 3, fold change ≥ 1.8). c Represents genes with no significant change in expression level after ETP treatment ( n = 3). d Venn diagram showing commonly deregulated genes in groups with different ETP concentrations ( n = 3). e Represents deregulated miRNA markers ( n = 3)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques: Expressing

    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)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques: 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)

    Journal: Archives of Toxicology

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

    doi: 10.1007/s00204-018-2170-7

    Figure Lengend 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)

    Article Snippet: Chemicals Etoposide (E1383) was obtained from Sigma-Aldrich Chemie GmbH (Germany), Liproxstatin-1 (S7699) was obtained from Selleckchem (Germany), and Pifithrin-α hydrobromide (2653) was obtained from Tocris (United Kingdom).

    Techniques: Immunostaining, Staining, Transmission Electron Microscopy

    Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM camptothecin for 24 hr. Both floating and attached cells were

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Insig2 is associated with colon tumorigenesis and inhibits Bax-mediated apoptosis

    doi: 10.1002/ijc.23510

    Figure Lengend Snippet: Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM camptothecin for 24 hr. Both floating and attached cells were

    Article Snippet: A variety of agents including etoposide (ETO, Sigma), 5-fluorouracil (5-FU, American Pharmaceutical Partners), camptothecin, staurosporine (STS) and Actinomycin D (Sigma) were used to induce apoptosis in HCT116 cells transfected with pcDNA3.1(+) (mock) or pCMV-Insig2-Myc.

    Techniques: Clone Assay, Transfection, Cell Culture

    The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cells HCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (* P

    Journal: Oncotarget

    Article Title: miR-191 promotes tumorigenesis of human colorectal cancer through targeting C/EBPβ

    doi:

    Figure Lengend Snippet: The involvement of miR-191 in the 5-Fu-induced cell apoptotic pathway in HCT116 cells HCT116 cells were treated with various commonly-used chemotherapeutic drugs, including 5-fluorouracil (5-Fu, 10 μg/ml), indomethacin (Indo, 10 μg/ml), cisplatin (Cis, 10 μg/ml) and etoposide (Eto, 2 μM). (A) Cell viability was assessed by CCK8 assays. (B) RT-PCR analysis of the relative expression of miR-191. (C) 5-Fu down-regulated miR-191 in a dose-dependent manner. HCT116 cells were treated with the indicated concentration of 5-Fu for 48 hours, total mRNA was isolated, and the miR-191 level was analyzed by RT-PCR (* P

    Article Snippet: 5-Fluorouracil (5-Fu), indomethacin (Indo), cisplatin (Cis) and etoposide (Eto) were obtained from Sigma Chemical Co. (St. Louis, MO, USA)

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay, Isolation

    HSP47 expression affected ER-resident chaperone interactions. The expression of GRP78/Bip, CHOP/GADD135, full-length ATF6, cleaved ATF6, phospho-PERK, or IRE1 protein in NIH3T3 cells after treatment with GalNAc-bn for the indicated periods was examined by western blot analysis using anti-GRP78/Bip antibodies (A), anti-GADD153 (CHOP) antibodies (A), anti-ATF6 antibodies (B), anti-phosphoPERK antibodies (C), or anti-IRE1α antibodies (C). NIH3T3 cells treated with thapsigargin (Tg) were used as positive controls for these UPR-related molecule activation. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells; ATF6-fl, ATF6 full length; ATF6-c, ATF6 cleavage.

    Journal: PLoS ONE

    Article Title: The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

    doi: 10.1371/journal.pone.0069732

    Figure Lengend Snippet: HSP47 expression affected ER-resident chaperone interactions. The expression of GRP78/Bip, CHOP/GADD135, full-length ATF6, cleaved ATF6, phospho-PERK, or IRE1 protein in NIH3T3 cells after treatment with GalNAc-bn for the indicated periods was examined by western blot analysis using anti-GRP78/Bip antibodies (A), anti-GADD153 (CHOP) antibodies (A), anti-ATF6 antibodies (B), anti-phosphoPERK antibodies (C), or anti-IRE1α antibodies (C). NIH3T3 cells treated with thapsigargin (Tg) were used as positive controls for these UPR-related molecule activation. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells; ATF6-fl, ATF6 full length; ATF6-c, ATF6 cleavage.

    Article Snippet: The chemical reagents used in this study included benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-bn), thapsigargin (Tg), tunicamycin(TM), staurosporine (STS), etoposide (Eto), and monensin (Sigma Chemical Co., St. Louis, MO, USA).

    Techniques: Expressing, Western Blot, Activation Assay, Transfection

    HSP47 expression affected mitochondria caspase-9 cleavage. (A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using an antibody against caspase-9. NIH3T3 cells treated with staurosporine (STS) were used as a positive control for caspase-9 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells. (B) CAG and BCL cells treated with STS for 12 h were examined by western blot analysis using an antibody against caspase-9. (C) Western blot analysis showed caspase-2 and GAPDH protein expression 2 d after transfection with caspase-2 siRNA. (D) CAG, BCL, and caspase-2 siRNA transfected NIH3T3 (Cas2 siRNA) cells treated with GalNAc-bn for 24 h were examined by western blot analysis using an antibody against caspase-9 after HSP47 siRNA transfection.

    Journal: PLoS ONE

    Article Title: The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

    doi: 10.1371/journal.pone.0069732

    Figure Lengend Snippet: HSP47 expression affected mitochondria caspase-9 cleavage. (A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using an antibody against caspase-9. NIH3T3 cells treated with staurosporine (STS) were used as a positive control for caspase-9 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells. (B) CAG and BCL cells treated with STS for 12 h were examined by western blot analysis using an antibody against caspase-9. (C) Western blot analysis showed caspase-2 and GAPDH protein expression 2 d after transfection with caspase-2 siRNA. (D) CAG, BCL, and caspase-2 siRNA transfected NIH3T3 (Cas2 siRNA) cells treated with GalNAc-bn for 24 h were examined by western blot analysis using an antibody against caspase-9 after HSP47 siRNA transfection.

    Article Snippet: The chemical reagents used in this study included benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-bn), thapsigargin (Tg), tunicamycin(TM), staurosporine (STS), etoposide (Eto), and monensin (Sigma Chemical Co., St. Louis, MO, USA).

    Techniques: Expressing, Western Blot, Positive Control, Transfection

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-16394-2

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

    Article Snippet: PLK1 inhibitors, GSK461364 and BI6727, ATM and ATR inhibitors, KU-55933 and VE-821, and etoposide were from Selleckchem.

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