anti γ h2ax Merck Kgaa Search Results


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  • 92
    Merck KGaA anti γ h2ax
    DNA damage after IR increased following treatment with RGD/P-AuNPs. Notes: ( A ) Representative confocal immunofluorescence images of <t>γ-H2AX</t> (red) in MDA-MB-231 cells after treatment with AuNPs and radiation (0 Gy or 4 Gy). Bar, 20 μm. ( B ) Number of γ-H2AX foci per nucleus in MDA-MB-231 cells was counted. Cells pre-cultured with AuNPs were fixed and stained with γ-H2AX antibody 12 h after IR (0 Gy or 4 Gy). At least 50 nuclei were counted in each independent experiment. Columns, mean (n=3), bars, SE, * P
    Anti γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Merck KGaA mouse anti γ h2ax
    Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by etoposide (Etop) and mitoxantrone (Mtx). (A) NB4 cells were treated with the indicated concentrations of etoposide. TOP2A and TOP2B complexes were quantified on a cell-by-cell basis by the TARDIS assay using anti-TOP2A (4566) or anti-TOP2B (4555), respectively. Data are displayed as scatterplots, each dot representing the integrated immunofluorescent signal from a single nucleus. Medians and interquartile ranges are indicated. The number of nuclei quantified for each condition are indicated above each plot. For TOP2A, fluorescent intensities were significantly above those of untreated cells for 1 µ M etoposide and above, whereas for TOP2B, significant increase was reached by 10 µ M. (B) Cells treated as in (A) were analyzed by immunofluorescence for phospho-S 139 histone H2AX ( γ H2AX). The results are displayed as the means of the medians of replicate experiments (number of replicas indicated above each bar) normalized to 100 μ M etoposide. Error bars represent the S.D. (C and D) NB4 cells were treated with the indicated concentrations of mitoxantrone and were analyzed for TOP2-DNA complexes and <t>γ</t> H2AX immunofluorescence as in (A) and (B), respectively. For both TOP2A and TOP2B, fluorescent intensities significantly increased compared with untreated cells at all concentrations of mitoxantrone from 0.1 to 20 µ M. (D) Significance values were determined using one-way ANOVA with Tukey correction for multiple comparisons. Significance values shown in blue refer to comparison with untreated cells. Error bars indicate S.D. values.
    Mouse Anti γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA anti γ h2ax ser139 antibody
    Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by etoposide (Etop) and mitoxantrone (Mtx). (A) NB4 cells were treated with the indicated concentrations of etoposide. TOP2A and TOP2B complexes were quantified on a cell-by-cell basis by the TARDIS assay using anti-TOP2A (4566) or anti-TOP2B (4555), respectively. Data are displayed as scatterplots, each dot representing the integrated immunofluorescent signal from a single nucleus. Medians and interquartile ranges are indicated. The number of nuclei quantified for each condition are indicated above each plot. For TOP2A, fluorescent intensities were significantly above those of untreated cells for 1 µ M etoposide and above, whereas for TOP2B, significant increase was reached by 10 µ M. (B) Cells treated as in (A) were analyzed by immunofluorescence for phospho-S 139 histone H2AX ( γ H2AX). The results are displayed as the means of the medians of replicate experiments (number of replicas indicated above each bar) normalized to 100 μ M etoposide. Error bars represent the S.D. (C and D) NB4 cells were treated with the indicated concentrations of mitoxantrone and were analyzed for TOP2-DNA complexes and <t>γ</t> H2AX immunofluorescence as in (A) and (B), respectively. For both TOP2A and TOP2B, fluorescent intensities significantly increased compared with untreated cells at all concentrations of mitoxantrone from 0.1 to 20 µ M. (D) Significance values were determined using one-way ANOVA with Tukey correction for multiple comparisons. Significance values shown in blue refer to comparison with untreated cells. Error bars indicate S.D. values.
    Anti γ H2ax Ser139 Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA 488 alex fluor anti γ h2ax antibody
    Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by etoposide (Etop) and mitoxantrone (Mtx). (A) NB4 cells were treated with the indicated concentrations of etoposide. TOP2A and TOP2B complexes were quantified on a cell-by-cell basis by the TARDIS assay using anti-TOP2A (4566) or anti-TOP2B (4555), respectively. Data are displayed as scatterplots, each dot representing the integrated immunofluorescent signal from a single nucleus. Medians and interquartile ranges are indicated. The number of nuclei quantified for each condition are indicated above each plot. For TOP2A, fluorescent intensities were significantly above those of untreated cells for 1 µ M etoposide and above, whereas for TOP2B, significant increase was reached by 10 µ M. (B) Cells treated as in (A) were analyzed by immunofluorescence for phospho-S 139 histone H2AX ( γ H2AX). The results are displayed as the means of the medians of replicate experiments (number of replicas indicated above each bar) normalized to 100 μ M etoposide. Error bars represent the S.D. (C and D) NB4 cells were treated with the indicated concentrations of mitoxantrone and were analyzed for TOP2-DNA complexes and <t>γ</t> H2AX immunofluorescence as in (A) and (B), respectively. For both TOP2A and TOP2B, fluorescent intensities significantly increased compared with untreated cells at all concentrations of mitoxantrone from 0.1 to 20 µ M. (D) Significance values were determined using one-way ANOVA with Tukey correction for multiple comparisons. Significance values shown in blue refer to comparison with untreated cells. Error bars indicate S.D. values.
    488 Alex Fluor Anti γ H2ax Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA anti γ h2ax rabbit polyclonal antibody
    (A) Western blotting analysis of wild-type and S260A-mutated XRCC4 in M10-derived transformants. Proliferating cell nuclear antigen (PCNA) is shown as the loading control. (B) Radiosensitivity of M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells measured by colony formation assay in 0.2% w/v agarose. Each symbol represents the average surviving fraction obtained from 8–10 repeated experiments, with the error bar indicating standard deviation. The P value was calculated by applying the data to Welch’s one-sided t -test. * P = 0.049, ** P = 0.023, *** P = 0.017. (C): Number of <t>γ-H2AX</t> foci per cell in M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells. Cells were harvested 4 h after 1 Gy irradiation or without irradiation and subjected to immunostaining using anti-γ-H2AX antibody and fluorescent-labeled secondary antibody. More than 60 cells were examined for each group. Each circle represents the number of foci in a single cell, and the horizontal bar shows the average number of foci per cell in the group. The P -value was calculated by applying the data to Welch’s one-sided t -test.
    Anti γ H2ax Rabbit Polyclonal Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA anti gamma h2ax ser139 antibody
    (A) Western blotting analysis of wild-type and S260A-mutated XRCC4 in M10-derived transformants. Proliferating cell nuclear antigen (PCNA) is shown as the loading control. (B) Radiosensitivity of M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells measured by colony formation assay in 0.2% w/v agarose. Each symbol represents the average surviving fraction obtained from 8–10 repeated experiments, with the error bar indicating standard deviation. The P value was calculated by applying the data to Welch’s one-sided t -test. * P = 0.049, ** P = 0.023, *** P = 0.017. (C): Number of <t>γ-H2AX</t> foci per cell in M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells. Cells were harvested 4 h after 1 Gy irradiation or without irradiation and subjected to immunostaining using anti-γ-H2AX antibody and fluorescent-labeled secondary antibody. More than 60 cells were examined for each group. Each circle represents the number of foci in a single cell, and the horizontal bar shows the average number of foci per cell in the group. The P -value was calculated by applying the data to Welch’s one-sided t -test.
    Anti Gamma H2ax Ser139 Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Merck KGaA monoclonal anti s139 γ h2ax
    Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and <t>anti-γ-H2AX</t> (green). Hoechst H33342 was used for chromosomal DNA staining.
    Monoclonal Anti S139 γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA γ h2ax antibody fitc conjugate
    Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and <t>anti-γ-H2AX</t> (green). Hoechst H33342 was used for chromosomal DNA staining.
    γ H2ax Antibody Fitc Conjugate, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck KGaA antibodies against γ h2ax fitc
    DSBs were analysed using the <t>γ-H2AX</t> assay ( A ) The induction of DSBs in R1 and SiHa was significantly higher after addition of PARP1- i to cDDP-based thermochermotherapy. In HeLa cells this was not found to be significant, although a trend is seen. R1: p = 0.048, SiHa: p = 0.035, HeLa: p = 0.068 From left to right: R1, SiHa, Hela cells. ( B ) One representative cell is depicted for each condition. Bars represent the mean of three independent experiments with the standard error of the mean (SEM). * p
    Antibodies Against γ H2ax Fitc, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse monoclonal antibody anti γ h2ax
    Higher levels of DNA damage were observed after treatment with a shorter time interval between ionizing radiation and hyperthermia. ( A ) Cells in S-phase were excluded to only count the <t>γ-H2AX</t> positive double strand breaks (DSBs) that were radiation-induced. ( B ) SiHa cells demonstrating the DNA damage using γ-H2AX foci—cells were fixed 24 h after treatments. ( C–E ) γ-H2AX foci of HPV16 + , HPV18 + , and HPV-negative cell lines between ionizing radiation (IR; 2 Gy) and hyperthermia (HT) after treatments with different time intervals, different sequences, and various temperatures. Means with standard deviation of at least three replicates are presented, with a minimum of 100 cells per replicate. * p
    Mouse Monoclonal Antibody Anti γ H2ax, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Merck KGaA mouse monoclonal anti phospho histone γ h2ax ser139
    Plk1 inhibitor treatment induces apoptosis in both subtypes of ASCs ASCs were subjected to Plk1 compounds (25 nM BI 2536, 25 nM BI 6727 or 25 μM Poloxin) for 4 days and 14 days. Media and compounds were renewed every 3 days. (A and B) To evaluate the induction of DNA damage, treated cells were stained for the DNA damage markers <t>γ-H2AX</t> and 53BP1, α-tubulin and DNA. Representatives are shown. White arrows indicate fragmented cell nuclei and red arrows depict abnormal enlarged cell nuclei. Scale: 25 μm. Insets are a four time magnification of boxed regions in leftist panels. Scale: 6.5 μm. ( C and D ) Quantification of γ-H2AX and 53BP1 double positive cells. The results are based on three independent experiments and presented as mean ± SEM. ** p
    Mouse Monoclonal Anti Phospho Histone γ H2ax Ser139, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 89/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA mouse anti human gamma h2ax antibody
    Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) <t>gamma-H2AX</t> (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.
    Mouse Anti Human Gamma H2ax Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA phosphorylated h2ax
    Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) <t>gamma-H2AX</t> (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.
    Phosphorylated H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Merck KGaA monoclonal antibodies against γ h2ax
    The yield of <t>γ-H2AX</t> foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.
    Monoclonal Antibodies Against γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA damage after IR increased following treatment with RGD/P-AuNPs. Notes: ( A ) Representative confocal immunofluorescence images of γ-H2AX (red) in MDA-MB-231 cells after treatment with AuNPs and radiation (0 Gy or 4 Gy). Bar, 20 μm. ( B ) Number of γ-H2AX foci per nucleus in MDA-MB-231 cells was counted. Cells pre-cultured with AuNPs were fixed and stained with γ-H2AX antibody 12 h after IR (0 Gy or 4 Gy). At least 50 nuclei were counted in each independent experiment. Columns, mean (n=3), bars, SE, * P

    Journal: International Journal of Nanomedicine

    Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

    doi: 10.2147/IJN.S137833

    Figure Lengend Snippet: DNA damage after IR increased following treatment with RGD/P-AuNPs. Notes: ( A ) Representative confocal immunofluorescence images of γ-H2AX (red) in MDA-MB-231 cells after treatment with AuNPs and radiation (0 Gy or 4 Gy). Bar, 20 μm. ( B ) Number of γ-H2AX foci per nucleus in MDA-MB-231 cells was counted. Cells pre-cultured with AuNPs were fixed and stained with γ-H2AX antibody 12 h after IR (0 Gy or 4 Gy). At least 50 nuclei were counted in each independent experiment. Columns, mean (n=3), bars, SE, * P

    Article Snippet: The following primary antibodies were used for immunofluorescence staining: anti-α5-integrin (Cell Signaling Technology), anti-αv-integrin (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology), anti-EEA1 (Cell Signaling Technology), anti-Rab5 (Cell Signaling Technology), anti-Rab7 (Cell Signaling Technology), anti-Rab9 (Cell Signaling Technology) and anti-γ-H2AX (Merck Millipore).

    Techniques: Immunofluorescence, Multiple Displacement Amplification, Cell Culture, Staining

    Histopathological analysis of H460 tumors following combination treatment with oridonin and radiation. Hematoxylin and eosin (H-E) staining and immunohistochemistry for cleaved caspase-3 and γ-H2AX were performed on tumors harvested at 14 days after IR. Representative images of H-E-stained tumors ( upper images ) and cleaved caspase-3- and γ-H2AX-positive cells ( middle images , brown staining) and quantification of cleaved caspase-3 and γ-H2AX-positive staining with six mice in each group ( lower plots , means ± SEM) are shown; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells

    doi: 10.3390/ijms19082378

    Figure Lengend Snippet: Histopathological analysis of H460 tumors following combination treatment with oridonin and radiation. Hematoxylin and eosin (H-E) staining and immunohistochemistry for cleaved caspase-3 and γ-H2AX were performed on tumors harvested at 14 days after IR. Representative images of H-E-stained tumors ( upper images ) and cleaved caspase-3- and γ-H2AX-positive cells ( middle images , brown staining) and quantification of cleaved caspase-3 and γ-H2AX-positive staining with six mice in each group ( lower plots , means ± SEM) are shown; * p

    Article Snippet: The specific antibodies were cleaved caspase-3 (1:1000, #9661, Cell Signaling Technology, Inc., Danvers, MA, USA), γ-H2AX (1:3000, 05-636, Merck KGaA, Darmstadt, Germany), and β-actin (1:5000, sc-47778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Staining, Immunohistochemistry, Mouse Assay

    Induction of γ-H2AX following Olaparib treatment. Induction of γ-H2AX foci was assessed 24 48 hrs after treatment with 10 μM Olaparib. γ-H2AX levels were quantified using flow cytometry and are depicted as Mean Fluorescent Intensity (MFI) normalised to vehicle for each cell line. Data are representative of at least 3 independent experiments (ANOVA and Tukey post test, * P

    Journal: PLoS ONE

    Article Title: Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus

    doi: 10.1371/journal.pone.0207934

    Figure Lengend Snippet: Induction of γ-H2AX following Olaparib treatment. Induction of γ-H2AX foci was assessed 24 48 hrs after treatment with 10 μM Olaparib. γ-H2AX levels were quantified using flow cytometry and are depicted as Mean Fluorescent Intensity (MFI) normalised to vehicle for each cell line. Data are representative of at least 3 independent experiments (ANOVA and Tukey post test, * P

    Article Snippet: Cell cycle and γ-H2AX Cell cycle distribution and levels of γ-H2AX (to quantify DNA DSB) were determined in prepared nuclei by flow cytometry using DAPI (Sigma-Aldrich, UK) and anti-γ-H2AX antibody (clone JBW301) fluorescein isothiocyanate (FITC) conjugate (Merck-Millipore, Watford, UK; catalogue 16-202A) using published methodology [ ].

    Techniques: Flow Cytometry, Cytometry

    Synergistic effects of the combination of trabectedin and olaparib on poly(ADP-ribosyl)ation and DNA damage in four different breast cancer cell lines. MCF7, MDA-MB-231, MDA-MB-436, and HCC-1937 cells were treated with the indicated concentrations of trabectedin, olaparib or the combination of both drugs for 24 hours. Then, each cell line was lysed and analyzed by western blot with anti-PAR and γ-H2AX antibodies. α-Tubulin was used as loading control. NT=non-treated cells; C1=combination of 0.05 nM trabectedin and 5 µM olaparib; C2=combination of 0.1 nM trabectedin and 5 µM olaparib; C3=combination of 0.5 nM trabectedin and 5 µM olaparib.

    Journal: Journal of Breast Cancer

    Article Title: Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines

    doi: 10.4048/jbc.2015.18.4.329

    Figure Lengend Snippet: Synergistic effects of the combination of trabectedin and olaparib on poly(ADP-ribosyl)ation and DNA damage in four different breast cancer cell lines. MCF7, MDA-MB-231, MDA-MB-436, and HCC-1937 cells were treated with the indicated concentrations of trabectedin, olaparib or the combination of both drugs for 24 hours. Then, each cell line was lysed and analyzed by western blot with anti-PAR and γ-H2AX antibodies. α-Tubulin was used as loading control. NT=non-treated cells; C1=combination of 0.05 nM trabectedin and 5 µM olaparib; C2=combination of 0.1 nM trabectedin and 5 µM olaparib; C3=combination of 0.5 nM trabectedin and 5 µM olaparib.

    Article Snippet: Antibodies against γ-H2AX (05-636), PAR (#551813), XPF (MS-1381), and XPG (A301-484A) were obtained from Merck Millipore (Billerica, USA), BD Pharmigen (San Jose, USA), NeoMarkers (Fremont, USA), and Bethyl Lab (Montgomery, USA), respectively.

    Techniques: Multiple Displacement Amplification, Western Blot

    Ripley’s K statistics of γ-H2AX ( A ) and 53BP1 ( B ), MRE11 ( C ) and p-ATM ( D ) signal points locating in the nucleus (blue) or inside the γ-H2AX-defined DNA damage track (green).

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Ripley’s K statistics of γ-H2AX ( A ) and 53BP1 ( B ), MRE11 ( C ) and p-ATM ( D ) signal points locating in the nucleus (blue) or inside the γ-H2AX-defined DNA damage track (green).

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques:

    Fluorescence microscopy of γ-H2AX and DNA damage response proteins 53BP1, MRE11 and p-ATM in a single alpha-trajectory. ( A ) Wide-field overview images of human leukocyte nuclei stained with DAPI (blue), γ-H2AX and different proteins (53BP1, MRE11, p-ATM) showing typical regions of interests used for SMLM image acquisition. ( B ) Representative SMLM signal point density image of a leukocyte nucleus showing several large γ-H2AX super-foci linearly aligned along the trajectory of a single alpha particle. Slice thickness 500 nm. ( C ) Magnification and signal coordinate representation of red box in ( B ) showing that one γ-H2AX damage super-focus consists of substructures and smaller regions of high signal densities at the nanoscale.

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Fluorescence microscopy of γ-H2AX and DNA damage response proteins 53BP1, MRE11 and p-ATM in a single alpha-trajectory. ( A ) Wide-field overview images of human leukocyte nuclei stained with DAPI (blue), γ-H2AX and different proteins (53BP1, MRE11, p-ATM) showing typical regions of interests used for SMLM image acquisition. ( B ) Representative SMLM signal point density image of a leukocyte nucleus showing several large γ-H2AX super-foci linearly aligned along the trajectory of a single alpha particle. Slice thickness 500 nm. ( C ) Magnification and signal coordinate representation of red box in ( B ) showing that one γ-H2AX damage super-focus consists of substructures and smaller regions of high signal densities at the nanoscale.

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques: Fluorescence, Microscopy, Staining

    Quantitative analysis of γ-H2AX and 53BP1, MRE11 and p-ATM signals in damage tracks. ( A ) Relative frequency distribution of the DNA damage response (DDR) proteins along alpha tracks. The extension of a track was defined as the distance of the farthest signal points along the axis in a damage track signal number histogram (e.g., Figure 2 ). It appears that the average track length computed for γ-H2AX, 53BP1, MRE11 and p-ATM signals is similar, indicating that protein signals are distributed over the full extension of the damaged chromatin along the alpha particle trajectory. ( B ) Average ratios of DDR protein signal numbers relative to γ-H2AX signal numbers per average alpha track. In all experiments, γ-H2AX signal points were most frequent, followed in decreasing order by 53BP1 > MRE11 > p-ATM. ( C ) Ratio of signal point number abundance for γ-H2AX, 53BP1, MRE11 and p-ATM inside the average γ-H2AX alpha-track relative to signals over the nucleus. The signal numbers for γ-H2AX in different co-staining experiments were similar; thus, all γ-H2AX data were pooled for further single-color analyses. The average ratios of signal points detected inside the respective γ-H2AX damage track mask versus signal points detected over the whole nucleus show that most γ-H2AX signals are concentrated inside the damage track, which is less so for 53BP1, MRE11 and p-ATM. ( D ) Fraction of DDR protein signal points co-localizing with γ-H2AX signal points within a defined radius of 95 nm. 45% of 53BP1 and MRE11 signals in a track co-localize with γ-H2AX signals in a 95 nm radius, while only 21% of p-ATM signals showed co-localization. Error bars represent standard deviation.

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Quantitative analysis of γ-H2AX and 53BP1, MRE11 and p-ATM signals in damage tracks. ( A ) Relative frequency distribution of the DNA damage response (DDR) proteins along alpha tracks. The extension of a track was defined as the distance of the farthest signal points along the axis in a damage track signal number histogram (e.g., Figure 2 ). It appears that the average track length computed for γ-H2AX, 53BP1, MRE11 and p-ATM signals is similar, indicating that protein signals are distributed over the full extension of the damaged chromatin along the alpha particle trajectory. ( B ) Average ratios of DDR protein signal numbers relative to γ-H2AX signal numbers per average alpha track. In all experiments, γ-H2AX signal points were most frequent, followed in decreasing order by 53BP1 > MRE11 > p-ATM. ( C ) Ratio of signal point number abundance for γ-H2AX, 53BP1, MRE11 and p-ATM inside the average γ-H2AX alpha-track relative to signals over the nucleus. The signal numbers for γ-H2AX in different co-staining experiments were similar; thus, all γ-H2AX data were pooled for further single-color analyses. The average ratios of signal points detected inside the respective γ-H2AX damage track mask versus signal points detected over the whole nucleus show that most γ-H2AX signals are concentrated inside the damage track, which is less so for 53BP1, MRE11 and p-ATM. ( D ) Fraction of DDR protein signal points co-localizing with γ-H2AX signal points within a defined radius of 95 nm. 45% of 53BP1 and MRE11 signals in a track co-localize with γ-H2AX signals in a 95 nm radius, while only 21% of p-ATM signals showed co-localization. Error bars represent standard deviation.

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques: Staining, Standard Deviation

    Masking and track nanostructure obtained by γ-H2AX and DNA damage response protein (53BP1, MRE11 and p-ATM) signal tagging. ( A ) Visualization of representative γ-H2AX (top left) and DSB-associated protein (MRE11; bottom left) signal point densities (blurred with gauss filter with σ gauss = 50 nm. DBSCAN analysis of γ-H2AX signal points using ε = 200 nm and adjusted minimum point number of 25. Overlay of γ-H2AX (top middle) and protein signal densities (bottom middle) with corresponding γ-H2AX foci outline (red/green/blue) show the relative microscale distribution along the damage track. γ-H2AX (top-right) and protein (bottom-right) localization signals were masked with corresponding γ-H2AX foci areas. Where necessary, the alpha particle trajectory was approximated by a linear fit (white line) through the barycenter coordinates of each focus. The underlying signal point distribution of γ-H2AX and respective protein was calculated perpendicularly along the fit curve. ( B ) Histogram of signal number distribution of γ-H2AX (blue) and an example protein (green) inside γ-H2AX super-foci along an estimated alpha particle trajectory derived as shown in ( A ), with the first signal point along the linear trajectory being set to the distance value of 0. In this example, the start-to-end orientation of damage tracks was arranged arbitrarily. ( C ) Illustrations of localization density images of γ-H2AX damage tracks (masked with the corresponding γ-H2AX super-foci outlines) co-stained with 53BP1 (top), MRE11 (middle) and p-ATM (bottom) along the estimated alpha trajectory lines (white lines). ( D ) Signal number histogram plots along the masked particle tracks showing the signal numbers for each γ-H2AX/protein pair depicted in ( C ). Overall, 53BP1 signal numbers were comparable or in some cases even higher than γ-H2AX signal numbers (top), with the distribution of signal points along the particle track showing strong correlation between γ-H2AX and 53BP1. Signal numbers for MRE11 were significantly lower than for γ-H2AX. Again, small peak regions of high signal densities seemed to occur between MRE11 and γ-H2AX, thereby revealing the presence of smaller complex sub-structures. p-ATM signal points are sparsely distributed along the damage track, but co-occurred with regions dense in γ-H2AX signals.

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Masking and track nanostructure obtained by γ-H2AX and DNA damage response protein (53BP1, MRE11 and p-ATM) signal tagging. ( A ) Visualization of representative γ-H2AX (top left) and DSB-associated protein (MRE11; bottom left) signal point densities (blurred with gauss filter with σ gauss = 50 nm. DBSCAN analysis of γ-H2AX signal points using ε = 200 nm and adjusted minimum point number of 25. Overlay of γ-H2AX (top middle) and protein signal densities (bottom middle) with corresponding γ-H2AX foci outline (red/green/blue) show the relative microscale distribution along the damage track. γ-H2AX (top-right) and protein (bottom-right) localization signals were masked with corresponding γ-H2AX foci areas. Where necessary, the alpha particle trajectory was approximated by a linear fit (white line) through the barycenter coordinates of each focus. The underlying signal point distribution of γ-H2AX and respective protein was calculated perpendicularly along the fit curve. ( B ) Histogram of signal number distribution of γ-H2AX (blue) and an example protein (green) inside γ-H2AX super-foci along an estimated alpha particle trajectory derived as shown in ( A ), with the first signal point along the linear trajectory being set to the distance value of 0. In this example, the start-to-end orientation of damage tracks was arranged arbitrarily. ( C ) Illustrations of localization density images of γ-H2AX damage tracks (masked with the corresponding γ-H2AX super-foci outlines) co-stained with 53BP1 (top), MRE11 (middle) and p-ATM (bottom) along the estimated alpha trajectory lines (white lines). ( D ) Signal number histogram plots along the masked particle tracks showing the signal numbers for each γ-H2AX/protein pair depicted in ( C ). Overall, 53BP1 signal numbers were comparable or in some cases even higher than γ-H2AX signal numbers (top), with the distribution of signal points along the particle track showing strong correlation between γ-H2AX and 53BP1. Signal numbers for MRE11 were significantly lower than for γ-H2AX. Again, small peak regions of high signal densities seemed to occur between MRE11 and γ-H2AX, thereby revealing the presence of smaller complex sub-structures. p-ATM signal points are sparsely distributed along the damage track, but co-occurred with regions dense in γ-H2AX signals.

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques: Derivative Assay, Staining

    Quantification of cluster densities for 53BP1 and MRE11 inside the average γ-H2AX damage track. ( A ) Distribution of signal point clusters (red) for 53BP1 (top) and MRE11 (bottom) calculated with cluster parameters r cluster = 10 nm and minimum point numbers = 2. The retrieved clusters consisted of predominantly 2–5 signal points in chromatin regions of about 30 nm in diameter. The insets to the lower right display the track and nuclear outline. The X and Y axes give the signal point coordinates. ( B ) Boxplot representation of cluster densities for 53BP1 (top) and MRE11 (bottom) with red lines and green asterisks indicating the median and mean values, respectively. Clusters were quantified inside and outside for each γ-H2AX damage track in a given nucleus. It appears that the density of clusters peaks inside the track, when compared to the remaining nuclear area. Background correction (corr.) of cluster densities (clusters/µm 2 ) for the occurrence of pan-nuclear clusters outside the track revealed similar values as specified in Table 1 .

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Quantification of cluster densities for 53BP1 and MRE11 inside the average γ-H2AX damage track. ( A ) Distribution of signal point clusters (red) for 53BP1 (top) and MRE11 (bottom) calculated with cluster parameters r cluster = 10 nm and minimum point numbers = 2. The retrieved clusters consisted of predominantly 2–5 signal points in chromatin regions of about 30 nm in diameter. The insets to the lower right display the track and nuclear outline. The X and Y axes give the signal point coordinates. ( B ) Boxplot representation of cluster densities for 53BP1 (top) and MRE11 (bottom) with red lines and green asterisks indicating the median and mean values, respectively. Clusters were quantified inside and outside for each γ-H2AX damage track in a given nucleus. It appears that the density of clusters peaks inside the track, when compared to the remaining nuclear area. Background correction (corr.) of cluster densities (clusters/µm 2 ) for the occurrence of pan-nuclear clusters outside the track revealed similar values as specified in Table 1 .

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques:

    SMLM data point coordinates of γ-H2AX and 53BP1, MRE11 and p-ATM in alpha-trajectories. Representation of SMLM signal point positions in Cartesian coordinates over the whole nucleus for ( A ) γ-H2AX, ( B ) 53BP1, ( C ) MRE11, and ( D ) p-ATM. Following the masking procedure applied (c.f. Figure 2 A), SMLM signal point coordinates are differentially colored for those present in γ-H2AX-marked damage track mask (green) and those detected throughout the nucleus (blue). The small grey-boxed sub-regions are blown up for better display to the right.

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: SMLM data point coordinates of γ-H2AX and 53BP1, MRE11 and p-ATM in alpha-trajectories. Representation of SMLM signal point positions in Cartesian coordinates over the whole nucleus for ( A ) γ-H2AX, ( B ) 53BP1, ( C ) MRE11, and ( D ) p-ATM. Following the masking procedure applied (c.f. Figure 2 A), SMLM signal point coordinates are differentially colored for those present in γ-H2AX-marked damage track mask (green) and those detected throughout the nucleus (blue). The small grey-boxed sub-regions are blown up for better display to the right.

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques:

    Orientation of γ-H2AX damage tracks consisting of at least three differently sized super-foci. ( A ) Analysis of γ-H2AX damage tracks consisting of at least three separate super-foci of decreasing size ( n = 53) oriented from largest (L), to medium (M), to smallest (S). The signal points inside each focus were normalized against the total measured signal numbers. In cases with more than one focus in between the largest and the smallest focus along the damage track ( n = 11), the average signal points per cluster was calculated. The results are presented as boxplots for γ-H2AX together with the respective co-stained 53BP1, MRE11 and p-ATM protein tags. ( B ) Boxplot of the ratios of 53BP1, MRE11 and p-ATM signal numbers (top to bottom, respectively) against corresponding co-stained γ-H2AX signals in L, M and S foci as defined in (A). The relative γ-H2AX/protein signal ratios remain relatively constant for all cluster sizes.

    Journal: Cancers

    Article Title: Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

    doi: 10.3390/cancers11121877

    Figure Lengend Snippet: Orientation of γ-H2AX damage tracks consisting of at least three differently sized super-foci. ( A ) Analysis of γ-H2AX damage tracks consisting of at least three separate super-foci of decreasing size ( n = 53) oriented from largest (L), to medium (M), to smallest (S). The signal points inside each focus were normalized against the total measured signal numbers. In cases with more than one focus in between the largest and the smallest focus along the damage track ( n = 11), the average signal points per cluster was calculated. The results are presented as boxplots for γ-H2AX together with the respective co-stained 53BP1, MRE11 and p-ATM protein tags. ( B ) Boxplot of the ratios of 53BP1, MRE11 and p-ATM signal numbers (top to bottom, respectively) against corresponding co-stained γ-H2AX signals in L, M and S foci as defined in (A). The relative γ-H2AX/protein signal ratios remain relatively constant for all cluster sizes.

    Article Snippet: Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova).

    Techniques: Staining

    Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by etoposide (Etop) and mitoxantrone (Mtx). (A) NB4 cells were treated with the indicated concentrations of etoposide. TOP2A and TOP2B complexes were quantified on a cell-by-cell basis by the TARDIS assay using anti-TOP2A (4566) or anti-TOP2B (4555), respectively. Data are displayed as scatterplots, each dot representing the integrated immunofluorescent signal from a single nucleus. Medians and interquartile ranges are indicated. The number of nuclei quantified for each condition are indicated above each plot. For TOP2A, fluorescent intensities were significantly above those of untreated cells for 1 µ M etoposide and above, whereas for TOP2B, significant increase was reached by 10 µ M. (B) Cells treated as in (A) were analyzed by immunofluorescence for phospho-S 139 histone H2AX ( γ H2AX). The results are displayed as the means of the medians of replicate experiments (number of replicas indicated above each bar) normalized to 100 μ M etoposide. Error bars represent the S.D. (C and D) NB4 cells were treated with the indicated concentrations of mitoxantrone and were analyzed for TOP2-DNA complexes and γ H2AX immunofluorescence as in (A) and (B), respectively. For both TOP2A and TOP2B, fluorescent intensities significantly increased compared with untreated cells at all concentrations of mitoxantrone from 0.1 to 20 µ M. (D) Significance values were determined using one-way ANOVA with Tukey correction for multiple comparisons. Significance values shown in blue refer to comparison with untreated cells. Error bars indicate S.D. values.

    Journal: Molecular Pharmacology

    Article Title: Intercalating TOP2 Poisons Attenuate Topoisomerase Action at Higher Concentrations

    doi: 10.1124/mol.119.117259

    Figure Lengend Snippet: Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by etoposide (Etop) and mitoxantrone (Mtx). (A) NB4 cells were treated with the indicated concentrations of etoposide. TOP2A and TOP2B complexes were quantified on a cell-by-cell basis by the TARDIS assay using anti-TOP2A (4566) or anti-TOP2B (4555), respectively. Data are displayed as scatterplots, each dot representing the integrated immunofluorescent signal from a single nucleus. Medians and interquartile ranges are indicated. The number of nuclei quantified for each condition are indicated above each plot. For TOP2A, fluorescent intensities were significantly above those of untreated cells for 1 µ M etoposide and above, whereas for TOP2B, significant increase was reached by 10 µ M. (B) Cells treated as in (A) were analyzed by immunofluorescence for phospho-S 139 histone H2AX ( γ H2AX). The results are displayed as the means of the medians of replicate experiments (number of replicas indicated above each bar) normalized to 100 μ M etoposide. Error bars represent the S.D. (C and D) NB4 cells were treated with the indicated concentrations of mitoxantrone and were analyzed for TOP2-DNA complexes and γ H2AX immunofluorescence as in (A) and (B), respectively. For both TOP2A and TOP2B, fluorescent intensities significantly increased compared with untreated cells at all concentrations of mitoxantrone from 0.1 to 20 µ M. (D) Significance values were determined using one-way ANOVA with Tukey correction for multiple comparisons. Significance values shown in blue refer to comparison with untreated cells. Error bars indicate S.D. values.

    Article Snippet: Anti-mouse γ H2AX (catalog number 05-636) was obtained from Merck-Millipore.

    Techniques: Immunofluorescence

    TERT inhibition by BIBR increases DNA damage in TERT-positive cells. ( a ) TERT-positive 4134/Late, 4134/TERT+, BL41 and BL41/B95.8 cells and TERT-negative 4134/TERT- and U2OS cells exposed for 24 h to BIBR or to DMSO as control, were stained with γ H2AX to evaluate DNA damage and analyzed by flow cytometry. Panels from one representative experiment are shown. ( b ) Levels of γ H2AX MFI in BIBR- and DMSO-treated cells. Significant differences between values in BIBR-treated versus DMSO-treated cells are shown: * P

    Journal: Cell Death & Disease

    Article Title: Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

    doi: 10.1038/cddis.2016.425

    Figure Lengend Snippet: TERT inhibition by BIBR increases DNA damage in TERT-positive cells. ( a ) TERT-positive 4134/Late, 4134/TERT+, BL41 and BL41/B95.8 cells and TERT-negative 4134/TERT- and U2OS cells exposed for 24 h to BIBR or to DMSO as control, were stained with γ H2AX to evaluate DNA damage and analyzed by flow cytometry. Panels from one representative experiment are shown. ( b ) Levels of γ H2AX MFI in BIBR- and DMSO-treated cells. Significant differences between values in BIBR-treated versus DMSO-treated cells are shown: * P

    Article Snippet: To visualize DNA damage foci, slides were incubated for 1 h with a mouse monoclonal anti-γ H2AX antibody (1 : 1000, Merck Millipore, Darmstadt, Germany) in PBG buffer, followed by Alexa Fluor 488 anti-mouse secondary antibody (Thermo Fisher Scientific) in PBG buffer.

    Techniques: Inhibition, Staining, Flow Cytometry, Cytometry

    (A) Western blotting analysis of wild-type and S260A-mutated XRCC4 in M10-derived transformants. Proliferating cell nuclear antigen (PCNA) is shown as the loading control. (B) Radiosensitivity of M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells measured by colony formation assay in 0.2% w/v agarose. Each symbol represents the average surviving fraction obtained from 8–10 repeated experiments, with the error bar indicating standard deviation. The P value was calculated by applying the data to Welch’s one-sided t -test. * P = 0.049, ** P = 0.023, *** P = 0.017. (C): Number of γ-H2AX foci per cell in M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells. Cells were harvested 4 h after 1 Gy irradiation or without irradiation and subjected to immunostaining using anti-γ-H2AX antibody and fluorescent-labeled secondary antibody. More than 60 cells were examined for each group. Each circle represents the number of foci in a single cell, and the horizontal bar shows the average number of foci per cell in the group. The P -value was calculated by applying the data to Welch’s one-sided t -test.

    Journal: Journal of Radiation Research

    Article Title: In cellulo phosphorylation of DNA double-strand break repair protein XRCC4 on Ser260 by DNA-PK

    doi: 10.1093/jrr/rry072

    Figure Lengend Snippet: (A) Western blotting analysis of wild-type and S260A-mutated XRCC4 in M10-derived transformants. Proliferating cell nuclear antigen (PCNA) is shown as the loading control. (B) Radiosensitivity of M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells measured by colony formation assay in 0.2% w/v agarose. Each symbol represents the average surviving fraction obtained from 8–10 repeated experiments, with the error bar indicating standard deviation. The P value was calculated by applying the data to Welch’s one-sided t -test. * P = 0.049, ** P = 0.023, *** P = 0.017. (C): Number of γ-H2AX foci per cell in M10-XRCC4 WT , M10-XRCC4 S320A and M10-CMV cells. Cells were harvested 4 h after 1 Gy irradiation or without irradiation and subjected to immunostaining using anti-γ-H2AX antibody and fluorescent-labeled secondary antibody. More than 60 cells were examined for each group. Each circle represents the number of foci in a single cell, and the horizontal bar shows the average number of foci per cell in the group. The P -value was calculated by applying the data to Welch’s one-sided t -test.

    Article Snippet: Slides were incubated first with 5% bovine serum albumin–containing PBS(–) (BSA-PBS) for 1 h at room temperature for blocking and then with anti-γ-H2AX rabbit polyclonal antibody (Merck Millipore, 05–636) in BSA-PBS at 4°C overnight.

    Techniques: Western Blot, Derivative Assay, Colony Assay, Standard Deviation, Irradiation, Immunostaining, Labeling

    Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and anti-γ-H2AX (green). Hoechst H33342 was used for chromosomal DNA staining.

    Journal: Oncotarget

    Article Title: A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    doi:

    Figure Lengend Snippet: Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and anti-γ-H2AX (green). Hoechst H33342 was used for chromosomal DNA staining.

    Article Snippet: Monoclonal anti-S139-γ-H2AX (clone JBW103) antibody was from Upstate (Merck-Millipore, France).

    Techniques: Irradiation, In Situ, Immunofluorescence, Labeling, Staining

    Function of the phosphorylated form of Ku70 in cell cycle checkpoint control, DNA repair and genomic stability ZR75.1 breast cancer cells express either wt-Ku70 (S27-S33-Ku70) or mutated Ku70 (A27-A33-K70 or E27-E33-Ku70). A. pS27-Ku70 expression was monitored using a pS27-Ku70 antibody. B. The cell cycle was assayed at different timepoints after IR (4Gy) in S27-S33-Ku70- (blue) and A27-A33-Ku70- (red) expressing cells E27-E33-Ku70 expressing cells shown cell cycle profile similar to S27-S33-Ku70 (not shown). The cell cycle profiles shown are of untreated cells (a), and of post-irradiated cells at 12 h (b), 24 h (c) and 48 h (d). C. Cell incorporation of ethynyl deoxyuridine (EdU) in S phase. Untreated (NT) or 12 h post-irradiated (4Gy) cells are shown. Cells expressed S27-S33-Ku70 (a, b) or A27-A33-Ku70 (c, d). D. Role of phospho-Ku70 in cell growth and proliferation following irradiation stress. The cell index (i.e. cell growth, proliferation and membrane potential) following IR (4 Gy) was defined using an xCELLigence® living cells’ real-time follow-up system (see Methods section). Full lines represent untreated cells and dashed lines indicate irradiated cells. A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells were evaluated. The cell index is given in arbitrary units (AU). E. Western blot analysis of γ-H2AX protein level following 2Gy irradiation. Cells expressing S27-S33-Ku70, A27-A33-Ku70 were untreated (NT) or harvested after indicated time post-irradiation. After PAGE of total protein extracts and transfer, the membranes were probed with anti-phospho-S139-H2AX and anti-β-actin antibodies. F. Kinetic of DNA damage-induced γ-H2AX, foci formation in untreated (NT) or irradiated (2Gy) cells expressing wild-type S27-S33-Ku70, mutated A27-A33-Ku70 or E27-E33-Ku70 at indicated time post-irradiation. G. γ-H2AX immunostaining results were analyzed in untreated and post-irradiated cells at the indicated timepoints in A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells. Cells exhibiting at least 5 foci per nuclei were included in this analysis. Counts were performed on at least 200 cells per condition and results are depicted as box plot distribution values [minimum (min), maximum (max), median, 25th and 75th percentiles (25th and 75th perc.)] of the number of foci obtained for each tested condition. Statistical analysis: a Wilcoxon rank test was performed. *** p

    Journal: Oncotarget

    Article Title: A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    doi:

    Figure Lengend Snippet: Function of the phosphorylated form of Ku70 in cell cycle checkpoint control, DNA repair and genomic stability ZR75.1 breast cancer cells express either wt-Ku70 (S27-S33-Ku70) or mutated Ku70 (A27-A33-K70 or E27-E33-Ku70). A. pS27-Ku70 expression was monitored using a pS27-Ku70 antibody. B. The cell cycle was assayed at different timepoints after IR (4Gy) in S27-S33-Ku70- (blue) and A27-A33-Ku70- (red) expressing cells E27-E33-Ku70 expressing cells shown cell cycle profile similar to S27-S33-Ku70 (not shown). The cell cycle profiles shown are of untreated cells (a), and of post-irradiated cells at 12 h (b), 24 h (c) and 48 h (d). C. Cell incorporation of ethynyl deoxyuridine (EdU) in S phase. Untreated (NT) or 12 h post-irradiated (4Gy) cells are shown. Cells expressed S27-S33-Ku70 (a, b) or A27-A33-Ku70 (c, d). D. Role of phospho-Ku70 in cell growth and proliferation following irradiation stress. The cell index (i.e. cell growth, proliferation and membrane potential) following IR (4 Gy) was defined using an xCELLigence® living cells’ real-time follow-up system (see Methods section). Full lines represent untreated cells and dashed lines indicate irradiated cells. A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells were evaluated. The cell index is given in arbitrary units (AU). E. Western blot analysis of γ-H2AX protein level following 2Gy irradiation. Cells expressing S27-S33-Ku70, A27-A33-Ku70 were untreated (NT) or harvested after indicated time post-irradiation. After PAGE of total protein extracts and transfer, the membranes were probed with anti-phospho-S139-H2AX and anti-β-actin antibodies. F. Kinetic of DNA damage-induced γ-H2AX, foci formation in untreated (NT) or irradiated (2Gy) cells expressing wild-type S27-S33-Ku70, mutated A27-A33-Ku70 or E27-E33-Ku70 at indicated time post-irradiation. G. γ-H2AX immunostaining results were analyzed in untreated and post-irradiated cells at the indicated timepoints in A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells. Cells exhibiting at least 5 foci per nuclei were included in this analysis. Counts were performed on at least 200 cells per condition and results are depicted as box plot distribution values [minimum (min), maximum (max), median, 25th and 75th percentiles (25th and 75th perc.)] of the number of foci obtained for each tested condition. Statistical analysis: a Wilcoxon rank test was performed. *** p

    Article Snippet: Monoclonal anti-S139-γ-H2AX (clone JBW103) antibody was from Upstate (Merck-Millipore, France).

    Techniques: Expressing, Irradiation, Western Blot, Polyacrylamide Gel Electrophoresis, Immunostaining

    DSBs were analysed using the γ-H2AX assay ( A ) The induction of DSBs in R1 and SiHa was significantly higher after addition of PARP1- i to cDDP-based thermochermotherapy. In HeLa cells this was not found to be significant, although a trend is seen. R1: p = 0.048, SiHa: p = 0.035, HeLa: p = 0.068 From left to right: R1, SiHa, Hela cells. ( B ) One representative cell is depicted for each condition. Bars represent the mean of three independent experiments with the standard error of the mean (SEM). * p

    Journal: Oncotarget

    Article Title: Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia

    doi: 10.18632/oncotarget.15922

    Figure Lengend Snippet: DSBs were analysed using the γ-H2AX assay ( A ) The induction of DSBs in R1 and SiHa was significantly higher after addition of PARP1- i to cDDP-based thermochermotherapy. In HeLa cells this was not found to be significant, although a trend is seen. R1: p = 0.048, SiHa: p = 0.035, HeLa: p = 0.068 From left to right: R1, SiHa, Hela cells. ( B ) One representative cell is depicted for each condition. Bars represent the mean of three independent experiments with the standard error of the mean (SEM). * p

    Article Snippet: After centrifugation, cells were stained with antibodies against γ-H2AX-FITC (4 μg/ml, Merck Millipore, USA).

    Techniques:

    The effects of PARP1- i , HT, cDDP and combined treatments on BRCA2, Rad51 and cell survival are shown ( A ) Western blots demonstrating R1, SiHa and HeLa cells are BRCA2 proficient. After HT, BRCA2 is downregulated. ( B ) γ-H2AX and Rad51 co-localization, to investigate activity of homologous recombination. ( C ) Clonogenic assays were performed to study the effect of the different treatment combinations, 10–12 days after treatments. The addition of PARP1- i to cDDP-based thermochemotherapy resulted in a significantly lower cell survival compared to cDDP-based thermochemotherapy alone. R1: p = 0.0008, SiHa: p = 0.034, HeLa: p = 0.021. The bar graph shows the mean of at least five independent experiments. From left to right: R1, SiHa, Hela cells. * p

    Journal: Oncotarget

    Article Title: Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia

    doi: 10.18632/oncotarget.15922

    Figure Lengend Snippet: The effects of PARP1- i , HT, cDDP and combined treatments on BRCA2, Rad51 and cell survival are shown ( A ) Western blots demonstrating R1, SiHa and HeLa cells are BRCA2 proficient. After HT, BRCA2 is downregulated. ( B ) γ-H2AX and Rad51 co-localization, to investigate activity of homologous recombination. ( C ) Clonogenic assays were performed to study the effect of the different treatment combinations, 10–12 days after treatments. The addition of PARP1- i to cDDP-based thermochemotherapy resulted in a significantly lower cell survival compared to cDDP-based thermochemotherapy alone. R1: p = 0.0008, SiHa: p = 0.034, HeLa: p = 0.021. The bar graph shows the mean of at least five independent experiments. From left to right: R1, SiHa, Hela cells. * p

    Article Snippet: After centrifugation, cells were stained with antibodies against γ-H2AX-FITC (4 μg/ml, Merck Millipore, USA).

    Techniques: Western Blot, Activity Assay, Homologous Recombination

    Higher levels of DNA damage were observed after treatment with a shorter time interval between ionizing radiation and hyperthermia. ( A ) Cells in S-phase were excluded to only count the γ-H2AX positive double strand breaks (DSBs) that were radiation-induced. ( B ) SiHa cells demonstrating the DNA damage using γ-H2AX foci—cells were fixed 24 h after treatments. ( C–E ) γ-H2AX foci of HPV16 + , HPV18 + , and HPV-negative cell lines between ionizing radiation (IR; 2 Gy) and hyperthermia (HT) after treatments with different time intervals, different sequences, and various temperatures. Means with standard deviation of at least three replicates are presented, with a minimum of 100 cells per replicate. * p

    Journal: Cancers

    Article Title: Radiosensitization by Hyperthermia: The Effects of Temperature, Sequence, and Time Interval in Cervical Cell Lines

    doi: 10.3390/cancers12030582

    Figure Lengend Snippet: Higher levels of DNA damage were observed after treatment with a shorter time interval between ionizing radiation and hyperthermia. ( A ) Cells in S-phase were excluded to only count the γ-H2AX positive double strand breaks (DSBs) that were radiation-induced. ( B ) SiHa cells demonstrating the DNA damage using γ-H2AX foci—cells were fixed 24 h after treatments. ( C–E ) γ-H2AX foci of HPV16 + , HPV18 + , and HPV-negative cell lines between ionizing radiation (IR; 2 Gy) and hyperthermia (HT) after treatments with different time intervals, different sequences, and various temperatures. Means with standard deviation of at least three replicates are presented, with a minimum of 100 cells per replicate. * p

    Article Snippet: Then, cells were stained with a primary mouse monoclonal antibody anti-γ-H2AX (Millipore, Merck, KGaA, Darmstadt, Germany, dilution 1:100 in TNBS for 90 min at room temperature).

    Techniques: Standard Deviation

    Plk1 inhibitor treatment induces apoptosis in both subtypes of ASCs ASCs were subjected to Plk1 compounds (25 nM BI 2536, 25 nM BI 6727 or 25 μM Poloxin) for 4 days and 14 days. Media and compounds were renewed every 3 days. (A and B) To evaluate the induction of DNA damage, treated cells were stained for the DNA damage markers γ-H2AX and 53BP1, α-tubulin and DNA. Representatives are shown. White arrows indicate fragmented cell nuclei and red arrows depict abnormal enlarged cell nuclei. Scale: 25 μm. Insets are a four time magnification of boxed regions in leftist panels. Scale: 6.5 μm. ( C and D ) Quantification of γ-H2AX and 53BP1 double positive cells. The results are based on three independent experiments and presented as mean ± SEM. ** p

    Journal: Oncotarget

    Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

    doi: 10.18632/oncotarget.12482

    Figure Lengend Snippet: Plk1 inhibitor treatment induces apoptosis in both subtypes of ASCs ASCs were subjected to Plk1 compounds (25 nM BI 2536, 25 nM BI 6727 or 25 μM Poloxin) for 4 days and 14 days. Media and compounds were renewed every 3 days. (A and B) To evaluate the induction of DNA damage, treated cells were stained for the DNA damage markers γ-H2AX and 53BP1, α-tubulin and DNA. Representatives are shown. White arrows indicate fragmented cell nuclei and red arrows depict abnormal enlarged cell nuclei. Scale: 25 μm. Insets are a four time magnification of boxed regions in leftist panels. Scale: 6.5 μm. ( C and D ) Quantification of γ-H2AX and 53BP1 double positive cells. The results are based on three independent experiments and presented as mean ± SEM. ** p

    Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

    Techniques: Staining

    Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) gamma-H2AX (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

    doi: 10.1369/0022155418761351

    Figure Lengend Snippet: Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) gamma-H2AX (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Both sequential slides were incubated with 1% bovine serum albumin (BSA) in PBS for 20 min. On slide 1, a predilution of mouse anti-human gamma-H2AX antibody (05-636-I; Merck Millipore, Darmstadt, Germany) and rabbit anti-human pro-Spc antibody (AB3786; Merck Millipore) was applied.

    Techniques: Immunofluorescence, Staining, Confocal Microscopy

    Flow chart of methodological steps. The methods are subdivided in a telomere and gamma-H2AX staining phase and a second, postelution phase, which include the IF staining of specific cell markers. Integration of data from phase 1 and 2 was done by scanning coordinates. Abbreviations: IF, immunofluorescence; CC10, club cell-10; FISH, fluorescence in situ hybridization; aSMA, smooth muscle actin.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

    doi: 10.1369/0022155418761351

    Figure Lengend Snippet: Flow chart of methodological steps. The methods are subdivided in a telomere and gamma-H2AX staining phase and a second, postelution phase, which include the IF staining of specific cell markers. Integration of data from phase 1 and 2 was done by scanning coordinates. Abbreviations: IF, immunofluorescence; CC10, club cell-10; FISH, fluorescence in situ hybridization; aSMA, smooth muscle actin.

    Article Snippet: Both sequential slides were incubated with 1% bovine serum albumin (BSA) in PBS for 20 min. On slide 1, a predilution of mouse anti-human gamma-H2AX antibody (05-636-I; Merck Millipore, Darmstadt, Germany) and rabbit anti-human pro-Spc antibody (AB3786; Merck Millipore) was applied.

    Techniques: Flow Cytometry, Staining, Immunofluorescence, Fluorescence In Situ Hybridization, Fluorescence, In Situ Hybridization

    Antibody and FISH probe elution procedures are essential for the use of multiple fluorophores. Representative images of a patient with pulmonary fibrosis. Z-stacked LSCM pictures of (A) telomeres (red), (B) gamma-H2AX (yellow), and (C) overlay stains containing the AT2 cell–specific pro-Spc marker (green). (D–F) Magnifications of boxed areas in images A–C, respectively. (G and H) Telomere and gamma-H2AX channels after FISH probe and antibody elution, showing complete loss of cell nucleus–specific signal and persistent presence of autofluorescent collagen and elastin fibers. (I) DAPI DNA staining (blue) and slide coordinates were used to determine the location. (J, K) ImageJ Telometer images showing manually encircled cells of telomere and gamma-H2AX analyzed pictures. Both images were mirrored to (L), indicating pro-Spc (AT2) and caveolin-1 (AT1) specific cell markers, to identify cell types. White arrows indicate autofluorescent collagen and elastin fibers. Scale bars represent 10 µm. Abbreviations: FISH, fluorescence in situ hybridization; LSCM, laser scanning confocal microscopy; AT2, alveolar type 2 pneumocyte; DAPI, 4′,6-diamidino-2-phenylindole; AT1, alveolar type 1 pneumocyte.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

    doi: 10.1369/0022155418761351

    Figure Lengend Snippet: Antibody and FISH probe elution procedures are essential for the use of multiple fluorophores. Representative images of a patient with pulmonary fibrosis. Z-stacked LSCM pictures of (A) telomeres (red), (B) gamma-H2AX (yellow), and (C) overlay stains containing the AT2 cell–specific pro-Spc marker (green). (D–F) Magnifications of boxed areas in images A–C, respectively. (G and H) Telomere and gamma-H2AX channels after FISH probe and antibody elution, showing complete loss of cell nucleus–specific signal and persistent presence of autofluorescent collagen and elastin fibers. (I) DAPI DNA staining (blue) and slide coordinates were used to determine the location. (J, K) ImageJ Telometer images showing manually encircled cells of telomere and gamma-H2AX analyzed pictures. Both images were mirrored to (L), indicating pro-Spc (AT2) and caveolin-1 (AT1) specific cell markers, to identify cell types. White arrows indicate autofluorescent collagen and elastin fibers. Scale bars represent 10 µm. Abbreviations: FISH, fluorescence in situ hybridization; LSCM, laser scanning confocal microscopy; AT2, alveolar type 2 pneumocyte; DAPI, 4′,6-diamidino-2-phenylindole; AT1, alveolar type 1 pneumocyte.

    Article Snippet: Both sequential slides were incubated with 1% bovine serum albumin (BSA) in PBS for 20 min. On slide 1, a predilution of mouse anti-human gamma-H2AX antibody (05-636-I; Merck Millipore, Darmstadt, Germany) and rabbit anti-human pro-Spc antibody (AB3786; Merck Millipore) was applied.

    Techniques: Fluorescence In Situ Hybridization, Marker, Staining, Fluorescence, In Situ Hybridization, Confocal Microscopy

    Telomere and gamma-H2AX fluorescence measurements in AT1 and AT2 lung cells of a patient with a PARN mutation. (A) FISH-stained telomere signals are significantly higher in AT1 than in AT2 cells ( p

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

    doi: 10.1369/0022155418761351

    Figure Lengend Snippet: Telomere and gamma-H2AX fluorescence measurements in AT1 and AT2 lung cells of a patient with a PARN mutation. (A) FISH-stained telomere signals are significantly higher in AT1 than in AT2 cells ( p

    Article Snippet: Both sequential slides were incubated with 1% bovine serum albumin (BSA) in PBS for 20 min. On slide 1, a predilution of mouse anti-human gamma-H2AX antibody (05-636-I; Merck Millipore, Darmstadt, Germany) and rabbit anti-human pro-Spc antibody (AB3786; Merck Millipore) was applied.

    Techniques: Fluorescence, Mutagenesis, Fluorescence In Situ Hybridization, Staining

    Elevated concentrations of H 2 O 2 result in dose-dependent increase in gamma-H2AX intensity. Adenocarcinomic human alveolar basal epithelial (A549) cells were treated in four batches with 0–100–250–1000 µM H 2 O 2 respectively. Cells for cytospins were spun down on glass slides and stained using a gamma-H2AX antibody. Quantification of fluorescence showed that cells treated with 250- and 1000-µM H 2 O 2 had significant stronger gamma-H2AX signals than null-treated cells. Each dot represents a cell. Medians are indicated with horizontal bars. Values of p

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

    doi: 10.1369/0022155418761351

    Figure Lengend Snippet: Elevated concentrations of H 2 O 2 result in dose-dependent increase in gamma-H2AX intensity. Adenocarcinomic human alveolar basal epithelial (A549) cells were treated in four batches with 0–100–250–1000 µM H 2 O 2 respectively. Cells for cytospins were spun down on glass slides and stained using a gamma-H2AX antibody. Quantification of fluorescence showed that cells treated with 250- and 1000-µM H 2 O 2 had significant stronger gamma-H2AX signals than null-treated cells. Each dot represents a cell. Medians are indicated with horizontal bars. Values of p

    Article Snippet: Both sequential slides were incubated with 1% bovine serum albumin (BSA) in PBS for 20 min. On slide 1, a predilution of mouse anti-human gamma-H2AX antibody (05-636-I; Merck Millipore, Darmstadt, Germany) and rabbit anti-human pro-Spc antibody (AB3786; Merck Millipore) was applied.

    Techniques: Staining, Fluorescence

    The yield of γ-H2AX foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.

    Journal: International Journal of Molecular Sciences

    Article Title: Changes in the Number of Double-Strand DNA Breaks in Chinese Hamster V79 Cells Exposed to ?-Radiation with Different Dose Rates

    doi: 10.3390/ijms140713719

    Figure Lengend Snippet: The yield of γ-H2AX foci in Chinese hamster V79 cells exposed to γ-rays at high (400 mGy/min), medium (10 mGy/min) and low (1 mGy/min) dose rates.

    Article Snippet: Slides were incubated with monoclonal antibodies against γ-H2AX (Anti-phospho-Histone H2AX Rabbit Monoclonal, Merck-Millipore, Darmstadt, Germany) at 4 °C over night.

    Techniques: