etoposide Search Results


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  • 99
    Millipore etoposide
    Effects of small molecules on the cell cycle. ( a , b ) Profiles of RF/GF ratio of FUCCI-HeLa after treatment of Bleomycin (BLM: 5 µg ml −1 ) or Camptothecin (CPT: 500 nM), Thymidine (2.5 mM), 5-Fluorouracil (5-FU: 1 µM), <t>Etoposide</t> (Etop: 500 nM), Nocodazole (Noc: 50 ng ml −1 ) ( a ) and MLN8237 (200 nM) ( b ) after release from G1/S compared to the control are shown. Time frame for one cell cycle in RF/GF ratio profile (black bar, left). Average time frame of one cell cycle of control (Cont) or MLN8237 (200 nM) treatment is graphically presented (right). ( c ) Representative fluorescent images from DMSO control (Cont) or 200 nM of MLN8237 treatment (MLN8237) at indicated time after G1/S release (minutes); white arrows indicate the representative cell, measured for time frame of cell cycle (left). Graphical presentation of time frame of G1 phase or S/G2/M phase of control (Cont) and MLN8237 treatment (MLN8237) ( N = 11) (right).
    Etoposide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/etoposide/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2021-06
    99/100 stars
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    99
    Selleck Chemicals etoposide
    Effect of ZLDI-8 on cytotoxic activity of <t>Etoposide</t> or paclitaxel on MHCC97-H cell-cycle arrest. Cells, were pre-treated with ZLDI-8, were treated with IC 50 concentration of Etoposide ( a ) or Paclitaxel ( b ). Then, the cells were harvested and flow cytometer was performed. Results were shown as photographs or mean ± SD. * p
    Etoposide, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/etoposide/product/Selleck Chemicals
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2021-06
    99/100 stars
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    86
    Bristol Myers etoposide
    <t>Etoposide-induced</t> RPA2 foci do not colocalize with replication factories. HeLa cells were treated for 1 h with 100 μM VP-16. Cells were stained with the polyclonal antibody to hLigI (a), with the polyclonal antibody to PCNA (b), and with the 9H8 mAb to RPA2 (c). Antibodies were revealed with a Cy5-conjugated secondary antibody (red). In the same panels sites of BrdU incorporation were revealed by the FITC-conjugated anti- BrdU mAb (green). Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of protein-BrdU colocalization. d, cells were costained with the PC10 mAb to PCNA (green) and the polyclonal antibody to hLigI (red). e, cells were costained with the 9H8 mAb to RPA2 (green) and the polyclonal antibody to PCNA (red). f, cells were costained with the 9H8 mAb to RPA2 (green) and with the polyclonal antibody to hLigI (red). mAbs were revealed with the FITC-conjugated anti-mouse secondary antibody, and polyclonal antibodies were revealed with the Cy5-conjugated anti-rabbit secondary antibody. Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of colocalization between the different proteins.
    Etoposide, supplied by Bristol Myers, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/etoposide/product/Bristol Myers
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2021-06
    86/100 stars
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    98
    Thermo Fisher etoposide
    Clonogenic survival assays after genotoxic treatments. Clonogenic survival assays of WT , TRIM29 −/−/−/+ , Ku70 −/− , PALB2 −/− , BRCA1 −/− , and REV1 −/− cells against <t>etoposide</t> (A), camptothecin (B), cisplatin (C), olaparib (D), and UV‐C (E) treatments. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, percent survival of WT cells versus TRIM29 −/−/−/+ cells, Student’s t ‐test).
    Etoposide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/etoposide/product/Thermo Fisher
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2021-06
    98/100 stars
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    N/A
    Topoisomerase II inhibitor
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    N/A
    Etoposide is a plant alkaloid and an inhibitor of topoisomerase II IC 60 3 μM It inhibits proliferation of a variety of adenocarcinoma cells ICs 0 005 12 200 µM
      Buy from Supplier


    N/A
    α Etoposide is a cytotoxic agent which belongs to the drug type topoisomerase inhibitor It is also an antineoplastic drug
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    Image Search Results


    Effects of small molecules on the cell cycle. ( a , b ) Profiles of RF/GF ratio of FUCCI-HeLa after treatment of Bleomycin (BLM: 5 µg ml −1 ) or Camptothecin (CPT: 500 nM), Thymidine (2.5 mM), 5-Fluorouracil (5-FU: 1 µM), Etoposide (Etop: 500 nM), Nocodazole (Noc: 50 ng ml −1 ) ( a ) and MLN8237 (200 nM) ( b ) after release from G1/S compared to the control are shown. Time frame for one cell cycle in RF/GF ratio profile (black bar, left). Average time frame of one cell cycle of control (Cont) or MLN8237 (200 nM) treatment is graphically presented (right). ( c ) Representative fluorescent images from DMSO control (Cont) or 200 nM of MLN8237 treatment (MLN8237) at indicated time after G1/S release (minutes); white arrows indicate the representative cell, measured for time frame of cell cycle (left). Graphical presentation of time frame of G1 phase or S/G2/M phase of control (Cont) and MLN8237 treatment (MLN8237) ( N = 11) (right).

    Journal: Royal Society Open Science

    Article Title: Screening of cytotoxic or cytostatic flavonoids with quantitative Fluorescent Ubiquitination-based Cell Cycle Indicator-based cell cycle assay

    doi: 10.1098/rsos.181303

    Figure Lengend Snippet: Effects of small molecules on the cell cycle. ( a , b ) Profiles of RF/GF ratio of FUCCI-HeLa after treatment of Bleomycin (BLM: 5 µg ml −1 ) or Camptothecin (CPT: 500 nM), Thymidine (2.5 mM), 5-Fluorouracil (5-FU: 1 µM), Etoposide (Etop: 500 nM), Nocodazole (Noc: 50 ng ml −1 ) ( a ) and MLN8237 (200 nM) ( b ) after release from G1/S compared to the control are shown. Time frame for one cell cycle in RF/GF ratio profile (black bar, left). Average time frame of one cell cycle of control (Cont) or MLN8237 (200 nM) treatment is graphically presented (right). ( c ) Representative fluorescent images from DMSO control (Cont) or 200 nM of MLN8237 treatment (MLN8237) at indicated time after G1/S release (minutes); white arrows indicate the representative cell, measured for time frame of cell cycle (left). Graphical presentation of time frame of G1 phase or S/G2/M phase of control (Cont) and MLN8237 treatment (MLN8237) ( N = 11) (right).

    Article Snippet: Luteolin (#L9283), apigenin (#10798), quercetin (#Q4951), chrysin (#C80105), propidium iodine (#P4170), nocodazole (#M1404), etoposide (#E1383) and camptothecin (#C9911) were purchased from Sigma-Aldrich.

    Techniques: Cycling Probe Technology

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

    Journal: Cell Death & Disease

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

    doi: 10.1038/s41419-018-0804-6

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

    Article Snippet: Anti-tumor agents, Sorafenib (Cat. No.: S7397), Paclitaxel (Cat. No.: S1150), and Etoposide (Cat. No.: S1225) were purchased from Selleck Corporation, Houston, Texas, USA.

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

    Etoposide-induced RPA2 foci do not colocalize with replication factories. HeLa cells were treated for 1 h with 100 μM VP-16. Cells were stained with the polyclonal antibody to hLigI (a), with the polyclonal antibody to PCNA (b), and with the 9H8 mAb to RPA2 (c). Antibodies were revealed with a Cy5-conjugated secondary antibody (red). In the same panels sites of BrdU incorporation were revealed by the FITC-conjugated anti- BrdU mAb (green). Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of protein-BrdU colocalization. d, cells were costained with the PC10 mAb to PCNA (green) and the polyclonal antibody to hLigI (red). e, cells were costained with the 9H8 mAb to RPA2 (green) and the polyclonal antibody to PCNA (red). f, cells were costained with the 9H8 mAb to RPA2 (green) and with the polyclonal antibody to hLigI (red). mAbs were revealed with the FITC-conjugated anti-mouse secondary antibody, and polyclonal antibodies were revealed with the Cy5-conjugated anti-rabbit secondary antibody. Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of colocalization between the different proteins.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: Etoposide-induced RPA2 foci do not colocalize with replication factories. HeLa cells were treated for 1 h with 100 μM VP-16. Cells were stained with the polyclonal antibody to hLigI (a), with the polyclonal antibody to PCNA (b), and with the 9H8 mAb to RPA2 (c). Antibodies were revealed with a Cy5-conjugated secondary antibody (red). In the same panels sites of BrdU incorporation were revealed by the FITC-conjugated anti- BrdU mAb (green). Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of protein-BrdU colocalization. d, cells were costained with the PC10 mAb to PCNA (green) and the polyclonal antibody to hLigI (red). e, cells were costained with the 9H8 mAb to RPA2 (green) and the polyclonal antibody to PCNA (red). f, cells were costained with the 9H8 mAb to RPA2 (green) and with the polyclonal antibody to hLigI (red). mAbs were revealed with the FITC-conjugated anti-mouse secondary antibody, and polyclonal antibodies were revealed with the Cy5-conjugated anti-rabbit secondary antibody. Confocal laser images of the same cell were taken and merged. Yellow spots indicate the extent of colocalization between the different proteins.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Staining, BrdU Incorporation Assay

    ATM is not necessary for the redistribution of RPA2 and hLigI induced by VP-16. (A) Total cell extracts were prepared from AT1, AT3, and HeLa cells, untreated (−) or grown for 1 h in 100 μM VP-16. Extracts were analyzed in Western blotting with 9H8 mAb to RPA2. (B) AT1 cells, untreated or grown for 1 h in 100 μM VP-16, were analyzed by indirect immunofluorescence with 9H8 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Conventional epifluorescence microscopy images of representative cell nuclei are shown. Identical results were observed with AT3 cells. (C) Exponentially growing AT1 cells were stained with 2B1 mAb to hLigI and with FITC-conjugated goat anti-mouse IgG secondary antibody. a, nucleus of a cell in G1; b–e, different patterns observed in nuclei of S-phase cells; f, nucleus of a cell in G2.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: ATM is not necessary for the redistribution of RPA2 and hLigI induced by VP-16. (A) Total cell extracts were prepared from AT1, AT3, and HeLa cells, untreated (−) or grown for 1 h in 100 μM VP-16. Extracts were analyzed in Western blotting with 9H8 mAb to RPA2. (B) AT1 cells, untreated or grown for 1 h in 100 μM VP-16, were analyzed by indirect immunofluorescence with 9H8 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Conventional epifluorescence microscopy images of representative cell nuclei are shown. Identical results were observed with AT3 cells. (C) Exponentially growing AT1 cells were stained with 2B1 mAb to hLigI and with FITC-conjugated goat anti-mouse IgG secondary antibody. a, nucleus of a cell in G1; b–e, different patterns observed in nuclei of S-phase cells; f, nucleus of a cell in G2.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Western Blot, Immunofluorescence, Epifluorescence Microscopy, Staining

    Staurosporine prevents the phosphorylation of RPA2 and the redistribution of RPA2 and hLigI. (A) Western blot analysis of 0.5% Triton X-100–insoluble cell extracts prepared from HeLa cells treated for 1 h with 100 μM VP-16 either in the presence (+) or in the absence (−) of 10 μM staurosporine. Staurosporine was added to the medium 15 min before VP-16. Untreated cells (c) were also analyzed. RPA2 was revealed with 9H8 mAb. (B) The histogram indicates the percentage of cells with RPA2 foci after a treatment of 1 h with VP-16 (100 μM), either in the absence (−) or in the presence (+) of 10 μM staurosporine. Error bars indicate the average error of the mean as determined from three separate experiments. (C) Immunofluorescence analysis of the subnuclear distribution of hLigI in untreated HeLa cells or in cells grown for 2 h in 100 μM VP-16–containing medium either in the absence (−) or in the presence (+) of 10 μM staurosporine. Cells were stained with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Arrows point to cells in which hLigI has a typical mid-S-phase pattern.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: Staurosporine prevents the phosphorylation of RPA2 and the redistribution of RPA2 and hLigI. (A) Western blot analysis of 0.5% Triton X-100–insoluble cell extracts prepared from HeLa cells treated for 1 h with 100 μM VP-16 either in the presence (+) or in the absence (−) of 10 μM staurosporine. Staurosporine was added to the medium 15 min before VP-16. Untreated cells (c) were also analyzed. RPA2 was revealed with 9H8 mAb. (B) The histogram indicates the percentage of cells with RPA2 foci after a treatment of 1 h with VP-16 (100 μM), either in the absence (−) or in the presence (+) of 10 μM staurosporine. Error bars indicate the average error of the mean as determined from three separate experiments. (C) Immunofluorescence analysis of the subnuclear distribution of hLigI in untreated HeLa cells or in cells grown for 2 h in 100 μM VP-16–containing medium either in the absence (−) or in the presence (+) of 10 μM staurosporine. Cells were stained with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Arrows point to cells in which hLigI has a typical mid-S-phase pattern.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Western Blot, Immunofluorescence, Staining

    VP-16 induces the disassembly of replication factories. (A) Exponentially growing HeLa cells were grown for the indicated time periods (hours) in the presence of 100 μM VP-16 and then immediately fixed. The distribution of hLigI was revealed with 2B1 mAb and with FITC-conjugated sheep anti-mouse IgG secondary antibody. At each time point we counted, in a pool of 500 randomly selected cells, the number of cells displaying hLigI in patterns typical of mid- and late S-phase (patterns III, IV, and V defined by O'Keefe et al. [1992]. The value measured at 1, 2, or 3 h of growth in the presence of VP-16 was expressed as the percentage of the number of cells with mid- and late S-phase patterns detected in control cells (0 h). Error bars indicate the average error of the mean as determined from three separate experiments. Flow cytometry analysis of the DNA content of asynchronously growing cells (Exp) and cells incubated for 3 h in the presence of 100 μM VP-16 is shown in the top. (B) Immunofluorescence analysis of HeLa cells untreated or incubated for 1 h in 100 μM VP-16 (1 h). Cells were stained either with 2B1 mAb to hLigI or with PC10 mAb to PCNA. FITC-conjugated goat anti-mouse IgG was used as a secondary antibody. For each point the confocal laser image of a single cell nucleus, displaying a mid-S-phase pattern, is shown to better visualize the dispersion of PCNA and hLigI from the replication factories triggered by VP-16.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: VP-16 induces the disassembly of replication factories. (A) Exponentially growing HeLa cells were grown for the indicated time periods (hours) in the presence of 100 μM VP-16 and then immediately fixed. The distribution of hLigI was revealed with 2B1 mAb and with FITC-conjugated sheep anti-mouse IgG secondary antibody. At each time point we counted, in a pool of 500 randomly selected cells, the number of cells displaying hLigI in patterns typical of mid- and late S-phase (patterns III, IV, and V defined by O'Keefe et al. [1992]. The value measured at 1, 2, or 3 h of growth in the presence of VP-16 was expressed as the percentage of the number of cells with mid- and late S-phase patterns detected in control cells (0 h). Error bars indicate the average error of the mean as determined from three separate experiments. Flow cytometry analysis of the DNA content of asynchronously growing cells (Exp) and cells incubated for 3 h in the presence of 100 μM VP-16 is shown in the top. (B) Immunofluorescence analysis of HeLa cells untreated or incubated for 1 h in 100 μM VP-16 (1 h). Cells were stained either with 2B1 mAb to hLigI or with PC10 mAb to PCNA. FITC-conjugated goat anti-mouse IgG was used as a secondary antibody. For each point the confocal laser image of a single cell nucleus, displaying a mid-S-phase pattern, is shown to better visualize the dispersion of PCNA and hLigI from the replication factories triggered by VP-16.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Flow Cytometry, Cytometry, Incubation, Immunofluorescence, Staining

    The effect of VP-16 on replication factories is dose dependent. Exponentially growing HeLa cells were treated for 3 h with different concentrations of VP-16 and analyzed in indirect immunofluorescence with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Confocal laser images were taken. a to f correspond, respectively, to cells treated with 0, 2, 10, 20, 50 and 100 μM VP-16. The arrows point to cells in which hLigI has a typical mid-S-phase pattern. Note the progressive disassembly of the replication factories.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: The effect of VP-16 on replication factories is dose dependent. Exponentially growing HeLa cells were treated for 3 h with different concentrations of VP-16 and analyzed in indirect immunofluorescence with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. Confocal laser images were taken. a to f correspond, respectively, to cells treated with 0, 2, 10, 20, 50 and 100 μM VP-16. The arrows point to cells in which hLigI has a typical mid-S-phase pattern. Note the progressive disassembly of the replication factories.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Immunofluorescence

    VP-16 induces the formation of RPA2 foci and the concomitant phosphorylation of the protein. (A) Exponentially growing HeLa cells, either untreated (−) or held for 1 h in 100 μM VP-16 (+) were stained with 9H8 mAb to RPA2 and with FITC-conjugated goat anti-mouse IgG secondary antibody. Conventional epifluorescence microscopy images of representative cell nuclei are shown. (B) Triton X-100–insoluble cell extract (0.5% ) was prepared from cells treated as above. Extracts were analyzed by Western blotting with 9H8 mAb. The arrow points to the hyperphosphorylated form of RPA2 detectable after VP-16 treatment.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: VP-16 induces the formation of RPA2 foci and the concomitant phosphorylation of the protein. (A) Exponentially growing HeLa cells, either untreated (−) or held for 1 h in 100 μM VP-16 (+) were stained with 9H8 mAb to RPA2 and with FITC-conjugated goat anti-mouse IgG secondary antibody. Conventional epifluorescence microscopy images of representative cell nuclei are shown. (B) Triton X-100–insoluble cell extract (0.5% ) was prepared from cells treated as above. Extracts were analyzed by Western blotting with 9H8 mAb. The arrow points to the hyperphosphorylated form of RPA2 detectable after VP-16 treatment.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Staining, Epifluorescence Microscopy, Western Blot

    Effect of aphidicolin and UV light on the subnuclear distribution of hLigI. Exponentially growing HeLa cells were stained after the indicated treatments with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. a, untreated cells; b, cells stained after 3 h from UV irradiation at 20 J/m 2 ; c, cells kept for 3 h in 2 μg/ml aphidicolin; d, cells treated for 3 h with 100 μM VP-16; e, cells grown for 3 h in 2 μg/ml aphidicolin and 100 μM VP-16. Arrows point to cells in which hLigI has a typical mid-S-phase pattern.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: Effect of aphidicolin and UV light on the subnuclear distribution of hLigI. Exponentially growing HeLa cells were stained after the indicated treatments with 2B1 mAb and with FITC-conjugated goat anti-mouse IgG secondary antibody. a, untreated cells; b, cells stained after 3 h from UV irradiation at 20 J/m 2 ; c, cells kept for 3 h in 2 μg/ml aphidicolin; d, cells treated for 3 h with 100 μM VP-16; e, cells grown for 3 h in 2 μg/ml aphidicolin and 100 μM VP-16. Arrows point to cells in which hLigI has a typical mid-S-phase pattern.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Staining, Irradiation

    VP-16 affects the phosphorylation status of hLigI and RPA2. (A) Exponentially growing HeLa cells were incubated for 3 h with 100 μM VP-16. After the drug was removed, cells were grown for an additional 3 h (3 + 3) or 24 h in complete medium. After 24 h of recovery, approximately half of the cells were still adherent to the plastic dish (3 + 24), whereas the others were floating in the medium (f). Total cell extracts were analyzed by Western blotting with the 1A4 mAb directed to phosphorylated Ser 66 of hLigI, a rabbit polyclonal antibody to hLigI, the 9H8 mAb to RPA2, and the C-2–10 mAb to PARP. We also analyzed a total cell extract prepared from untreated HeLa cells (c). The 85-kDa proteolytic fragment of PARP detectable in apoptotic cells is indicated (arrow). (B) Exponentially growing HeLa cells were treated with 100 μM VP-16. At the indicated times (hours) total cell extracts were analyzed by Western blotting with 9H8 mAb to RPA2 and 1A4 mAb to hLigI.

    Journal: Molecular Biology of the Cell

    Article Title: Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

    doi:

    Figure Lengend Snippet: VP-16 affects the phosphorylation status of hLigI and RPA2. (A) Exponentially growing HeLa cells were incubated for 3 h with 100 μM VP-16. After the drug was removed, cells were grown for an additional 3 h (3 + 3) or 24 h in complete medium. After 24 h of recovery, approximately half of the cells were still adherent to the plastic dish (3 + 24), whereas the others were floating in the medium (f). Total cell extracts were analyzed by Western blotting with the 1A4 mAb directed to phosphorylated Ser 66 of hLigI, a rabbit polyclonal antibody to hLigI, the 9H8 mAb to RPA2, and the C-2–10 mAb to PARP. We also analyzed a total cell extract prepared from untreated HeLa cells (c). The 85-kDa proteolytic fragment of PARP detectable in apoptotic cells is indicated (arrow). (B) Exponentially growing HeLa cells were treated with 100 μM VP-16. At the indicated times (hours) total cell extracts were analyzed by Western blotting with 9H8 mAb to RPA2 and 1A4 mAb to hLigI.

    Article Snippet: Cells were treated for the time periods indicated in the text with different concentrations of etoposide (Vepesid: Bristol-Myers Squibb, New York, NY).

    Techniques: Incubation, Western Blot

    Clonogenic survival assays after genotoxic treatments. Clonogenic survival assays of WT , TRIM29 −/−/−/+ , Ku70 −/− , PALB2 −/− , BRCA1 −/− , and REV1 −/− cells against etoposide (A), camptothecin (B), cisplatin (C), olaparib (D), and UV‐C (E) treatments. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, percent survival of WT cells versus TRIM29 −/−/−/+ cells, Student’s t ‐test).

    Journal: FEBS Open Bio

    Article Title: TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double‐strand breaks in vertebrate cells

    doi: 10.1002/2211-5463.12954

    Figure Lengend Snippet: Clonogenic survival assays after genotoxic treatments. Clonogenic survival assays of WT , TRIM29 −/−/−/+ , Ku70 −/− , PALB2 −/− , BRCA1 −/− , and REV1 −/− cells against etoposide (A), camptothecin (B), cisplatin (C), olaparib (D), and UV‐C (E) treatments. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, percent survival of WT cells versus TRIM29 −/−/−/+ cells, Student’s t ‐test).

    Article Snippet: Cells were harvested onto the surface of a glass slide at the indicated time points using a Cytospin4 cytocentrifuge (Thermo Fisher Scientific), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for 10 min, and blocked in Odyssey® blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The cells were then incubated with mouse monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:500; CST, cat. #80312, Danvers, MA, USA), and rabbit polyclonal anti‐53BP1 antibody (1:500; Novus Biologicals, cat. #NB100‐904, Centennial, CO, USA) for the etoposide‐induced foci formation assay, or rabbit monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:1000; CST, cat. #9718) and mouse monoclonal anti‐RAD51 antibody (1:200; Santa Cruz, cat. #sc‐398587, Dallas, TX, USA) for the camptothecin‐induced foci formation assay at room temperature for 1 h. After intensive washing, the cells were incubated with goat anti‐mouse IgG conjugated with Dylight 594 (1:500; Thermo Fisher Scientific, cat. #35510) and goat anti‐rabbit IgG conjugated with Dylight 488 (1:500; Thermo Fisher Scientific, cat. #35552), and donkey anti‐rabbit IgG conjugated with Alexa Fluor 594 (1:500; Thermo Fisher Scientific, cat. #A‐21207) and donkey anti‐mouse IgG conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific, cat. #A‐21202) for etoposide‐ and camptothecin‐induced foci formation assays, respectively, at room temperature for 1 h. Cells were counterstained with Hoechst 33258 (1:2000; Thermo Fisher Scientific), and foci of ɣ‐H2AX, 53BP1, and RAD51 were observed under a fluorescence microscope from Nikon (Eclipse Ci Series, Tokyo, Japan).

    Techniques:

    ɣ‐H2AX foci formation induced by 1 µ m etoposide treatment. 1 µ m etoposide was used to induce ɣ‐H2AX foci formation of each indicated genotype. The formation of ɣ‐H2AX foci was monitored at the indicated time points, shown by representative images (A) and quantification of ɣ‐H2AX‐positive cells, which contain more than four ɣ‐H2AX foci (B), and a number of the ɣ‐H2AX foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments. The significance in difference between two groups was tested by Student’s t ‐test. A scale bar, 10 µm. (Figures 3 and 4 represent a single, continuous experiment. The 2‐h time point of Fig. 3 is the same as 0‐h time point in Fig. 4 . Some images from certain time points are represented in both figures.)

    Journal: FEBS Open Bio

    Article Title: TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double‐strand breaks in vertebrate cells

    doi: 10.1002/2211-5463.12954

    Figure Lengend Snippet: ɣ‐H2AX foci formation induced by 1 µ m etoposide treatment. 1 µ m etoposide was used to induce ɣ‐H2AX foci formation of each indicated genotype. The formation of ɣ‐H2AX foci was monitored at the indicated time points, shown by representative images (A) and quantification of ɣ‐H2AX‐positive cells, which contain more than four ɣ‐H2AX foci (B), and a number of the ɣ‐H2AX foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments. The significance in difference between two groups was tested by Student’s t ‐test. A scale bar, 10 µm. (Figures 3 and 4 represent a single, continuous experiment. The 2‐h time point of Fig. 3 is the same as 0‐h time point in Fig. 4 . Some images from certain time points are represented in both figures.)

    Article Snippet: Cells were harvested onto the surface of a glass slide at the indicated time points using a Cytospin4 cytocentrifuge (Thermo Fisher Scientific), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for 10 min, and blocked in Odyssey® blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The cells were then incubated with mouse monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:500; CST, cat. #80312, Danvers, MA, USA), and rabbit polyclonal anti‐53BP1 antibody (1:500; Novus Biologicals, cat. #NB100‐904, Centennial, CO, USA) for the etoposide‐induced foci formation assay, or rabbit monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:1000; CST, cat. #9718) and mouse monoclonal anti‐RAD51 antibody (1:200; Santa Cruz, cat. #sc‐398587, Dallas, TX, USA) for the camptothecin‐induced foci formation assay at room temperature for 1 h. After intensive washing, the cells were incubated with goat anti‐mouse IgG conjugated with Dylight 594 (1:500; Thermo Fisher Scientific, cat. #35510) and goat anti‐rabbit IgG conjugated with Dylight 488 (1:500; Thermo Fisher Scientific, cat. #35552), and donkey anti‐rabbit IgG conjugated with Alexa Fluor 594 (1:500; Thermo Fisher Scientific, cat. #A‐21207) and donkey anti‐mouse IgG conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific, cat. #A‐21202) for etoposide‐ and camptothecin‐induced foci formation assays, respectively, at room temperature for 1 h. Cells were counterstained with Hoechst 33258 (1:2000; Thermo Fisher Scientific), and foci of ɣ‐H2AX, 53BP1, and RAD51 were observed under a fluorescence microscope from Nikon (Eclipse Ci Series, Tokyo, Japan).

    Techniques:

    Foci formation of 53BP1 in response to etoposide. 53BP1 foci formation of each genotype induced by 1 µ m etoposide was investigated at the indicated time points, shown by representative images (A) and quantification of 53BP1‐positive cells, which contain more than four 53BP1 foci (B), and a number of the 53BP1 foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, Student’s t ‐test). A scale bar, 10 µm.

    Journal: FEBS Open Bio

    Article Title: TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double‐strand breaks in vertebrate cells

    doi: 10.1002/2211-5463.12954

    Figure Lengend Snippet: Foci formation of 53BP1 in response to etoposide. 53BP1 foci formation of each genotype induced by 1 µ m etoposide was investigated at the indicated time points, shown by representative images (A) and quantification of 53BP1‐positive cells, which contain more than four 53BP1 foci (B), and a number of the 53BP1 foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, Student’s t ‐test). A scale bar, 10 µm.

    Article Snippet: Cells were harvested onto the surface of a glass slide at the indicated time points using a Cytospin4 cytocentrifuge (Thermo Fisher Scientific), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for 10 min, and blocked in Odyssey® blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The cells were then incubated with mouse monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:500; CST, cat. #80312, Danvers, MA, USA), and rabbit polyclonal anti‐53BP1 antibody (1:500; Novus Biologicals, cat. #NB100‐904, Centennial, CO, USA) for the etoposide‐induced foci formation assay, or rabbit monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:1000; CST, cat. #9718) and mouse monoclonal anti‐RAD51 antibody (1:200; Santa Cruz, cat. #sc‐398587, Dallas, TX, USA) for the camptothecin‐induced foci formation assay at room temperature for 1 h. After intensive washing, the cells were incubated with goat anti‐mouse IgG conjugated with Dylight 594 (1:500; Thermo Fisher Scientific, cat. #35510) and goat anti‐rabbit IgG conjugated with Dylight 488 (1:500; Thermo Fisher Scientific, cat. #35552), and donkey anti‐rabbit IgG conjugated with Alexa Fluor 594 (1:500; Thermo Fisher Scientific, cat. #A‐21207) and donkey anti‐mouse IgG conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific, cat. #A‐21202) for etoposide‐ and camptothecin‐induced foci formation assays, respectively, at room temperature for 1 h. Cells were counterstained with Hoechst 33258 (1:2000; Thermo Fisher Scientific), and foci of ɣ‐H2AX, 53BP1, and RAD51 were observed under a fluorescence microscope from Nikon (Eclipse Ci Series, Tokyo, Japan).

    Techniques:

    DSB repair kinetics of DNA DSBs induced by etoposide as measured by the dissolution of ɣ‐H2AX foci. ɣ‐H2AX foci formation induced by 1 µ m etoposide treatment for 2 h of the indicated genotypes. ɣ‐H2AX foci formation was investigated after the treatment at the indicated time points, shown by representative images (A) and quantification of ɣ‐H2AX‐positive cells, which contain more than four ɣ‐H2AX foci (B), and a number of the ɣ‐H2AX foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001, Student’s t ‐test). A scale bar, 10 µm. (Figures 3 and 4 represent a single, continuous experiment. The 2 h time point of Fig. 3 is the same as 0 h time point in Fig. 4 . Some images from certain time points are represented in both figures.)

    Journal: FEBS Open Bio

    Article Title: TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double‐strand breaks in vertebrate cells

    doi: 10.1002/2211-5463.12954

    Figure Lengend Snippet: DSB repair kinetics of DNA DSBs induced by etoposide as measured by the dissolution of ɣ‐H2AX foci. ɣ‐H2AX foci formation induced by 1 µ m etoposide treatment for 2 h of the indicated genotypes. ɣ‐H2AX foci formation was investigated after the treatment at the indicated time points, shown by representative images (A) and quantification of ɣ‐H2AX‐positive cells, which contain more than four ɣ‐H2AX foci (B), and a number of the ɣ‐H2AX foci per nucleus (C). Median values are indicated in red. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001, Student’s t ‐test). A scale bar, 10 µm. (Figures 3 and 4 represent a single, continuous experiment. The 2 h time point of Fig. 3 is the same as 0 h time point in Fig. 4 . Some images from certain time points are represented in both figures.)

    Article Snippet: Cells were harvested onto the surface of a glass slide at the indicated time points using a Cytospin4 cytocentrifuge (Thermo Fisher Scientific), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for 10 min, and blocked in Odyssey® blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The cells were then incubated with mouse monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:500; CST, cat. #80312, Danvers, MA, USA), and rabbit polyclonal anti‐53BP1 antibody (1:500; Novus Biologicals, cat. #NB100‐904, Centennial, CO, USA) for the etoposide‐induced foci formation assay, or rabbit monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:1000; CST, cat. #9718) and mouse monoclonal anti‐RAD51 antibody (1:200; Santa Cruz, cat. #sc‐398587, Dallas, TX, USA) for the camptothecin‐induced foci formation assay at room temperature for 1 h. After intensive washing, the cells were incubated with goat anti‐mouse IgG conjugated with Dylight 594 (1:500; Thermo Fisher Scientific, cat. #35510) and goat anti‐rabbit IgG conjugated with Dylight 488 (1:500; Thermo Fisher Scientific, cat. #35552), and donkey anti‐rabbit IgG conjugated with Alexa Fluor 594 (1:500; Thermo Fisher Scientific, cat. #A‐21207) and donkey anti‐mouse IgG conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific, cat. #A‐21202) for etoposide‐ and camptothecin‐induced foci formation assays, respectively, at room temperature for 1 h. Cells were counterstained with Hoechst 33258 (1:2000; Thermo Fisher Scientific), and foci of ɣ‐H2AX, 53BP1, and RAD51 were observed under a fluorescence microscope from Nikon (Eclipse Ci Series, Tokyo, Japan).

    Techniques: