Structured Review

Merck KGaA anti γ h2ax
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 <t>γ-H2AX</t> 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
Anti γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti γ h2ax/product/Merck KGaA
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti γ h2ax - by Bioz Stars, 2020-01
93/100 stars

Images

1) Product Images from "Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082378

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
Figure Legend 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

Techniques Used: Staining, Immunohistochemistry, Mouse Assay

2) Product Images from "Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082378

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
Figure Legend 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

Techniques Used: Staining, Immunohistochemistry, Mouse Assay

3) Product Images from "Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells"

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

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S137833

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
Figure Legend 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

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

4) Product Images from "Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe"

Article Title: Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.117

Depletion of Cdc5L results in kinetochore-microtubule attachment defects and DNA damage. Synchronized HeLa cells transfected with control or  Cdc5L  siRNA were treated with MG132 to prevent anaphase onset ( a – d ). Coverslips were chilled on ice for 10 min ( b ) or fixed immediately ( a ) and stained with anti-centromere antibody (ACA; for kinetochores; red), anti- α -tubulin antibody (microtubules; green) and SYTOX Blue nucleic acid stain (for DNA; blue), scale bar, 10  μ m. For spindle checkpoint analysis( c  and  d ), the cells were stained with anti-Bub1 or BubR1 antibody (green) and ACA (for kinetochores; red), scale bar, 10  μ m. ( e  and  f ) Quantification of Bub1 and BubR1 levels described as in  c  and  d  were analyzed. Bub1 and BubR1 signals were normalized to ACA. For Bub1 quantification, control-knockdown cells ( n =4) and Cdc5L-knockdown cells ( n =10); for BubR1 quantification, control-knockdown cells ( n =7) and Cdc5L-knockdown cells ( n =14). Data are shown as mean±S.D. ( g ) HeLa cells were transfected with control or Cdc5L siRNA, then stained with anti- γ -H2AX antibody (green) and PI (for DNA; red), scale bar, 10  μ m. ( h ) A box-and-whisker plot showing the number of  γ -H2AX foci per cell described as in  g  cells with control siRNA ( n =328) or  Cdc5L  siRNA ( n =276). Outliers are indicated by open circles, extremes by asterisks
Figure Legend Snippet: Depletion of Cdc5L results in kinetochore-microtubule attachment defects and DNA damage. Synchronized HeLa cells transfected with control or Cdc5L siRNA were treated with MG132 to prevent anaphase onset ( a – d ). Coverslips were chilled on ice for 10 min ( b ) or fixed immediately ( a ) and stained with anti-centromere antibody (ACA; for kinetochores; red), anti- α -tubulin antibody (microtubules; green) and SYTOX Blue nucleic acid stain (for DNA; blue), scale bar, 10  μ m. For spindle checkpoint analysis( c and d ), the cells were stained with anti-Bub1 or BubR1 antibody (green) and ACA (for kinetochores; red), scale bar, 10  μ m. ( e and f ) Quantification of Bub1 and BubR1 levels described as in c and d were analyzed. Bub1 and BubR1 signals were normalized to ACA. For Bub1 quantification, control-knockdown cells ( n =4) and Cdc5L-knockdown cells ( n =10); for BubR1 quantification, control-knockdown cells ( n =7) and Cdc5L-knockdown cells ( n =14). Data are shown as mean±S.D. ( g ) HeLa cells were transfected with control or Cdc5L siRNA, then stained with anti- γ -H2AX antibody (green) and PI (for DNA; red), scale bar, 10  μ m. ( h ) A box-and-whisker plot showing the number of γ -H2AX foci per cell described as in g cells with control siRNA ( n =328) or Cdc5L siRNA ( n =276). Outliers are indicated by open circles, extremes by asterisks

Techniques Used: Transfection, Staining, Whisker Assay

Related Articles

Centrifugation:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cell suspension was sonicated and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 10 min, the supernatants were collected and protein concentrations were quantified using the bicinchoninic acid (BCA) method (23227, Pierce Biotechnology, Inc., Waltham, MA, USA). .. 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).

Sonication:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cell suspension was sonicated and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 10 min, the supernatants were collected and protein concentrations were quantified using the bicinchoninic acid (BCA) method (23227, Pierce Biotechnology, Inc., Waltham, MA, USA). .. 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).

Radio Immunoprecipitation:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cells were trypsinized and lysed in 100 µL of ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.5% SDS, 1 mM Na3 VO4 , 5 mM NaF, and 1 mM phenylmethylsulfonyl fluoride). .. 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).

Immunohistochemistry:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: Paragraph title: 4.9. Immunohistochemistry and Quantification ... The sections were incubated with anti-cleaved caspase-3 (1:100, #9661, Cell Signaling Technology, Inc.), anti-γ-H2AX (1:100, 05-636, Merck KGaA), or anti-Ki-67 (1:200, DRM004, Acris Antibodies, Herford, Germany) antibodies at 4 °C overnight and then washed with PBS containing 0.05% Triton X-100.

Incubation:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cell suspension was sonicated and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 10 min, the supernatants were collected and protein concentrations were quantified using the bicinchoninic acid (BCA) method (23227, Pierce Biotechnology, Inc., Waltham, MA, USA). .. 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).

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: .. The sections were incubated with anti-cleaved caspase-3 (1:100, #9661, Cell Signaling Technology, Inc.), anti-γ-H2AX (1:100, 05-636, Merck KGaA), or anti-Ki-67 (1:200, DRM004, Acris Antibodies, Herford, Germany) antibodies at 4 °C overnight and then washed with PBS containing 0.05% Triton X-100. .. The sections were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min and counterstained with hematoxylin.

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Secondary antibodies (conjugated to a fluorophore) were diluted 1:1000 in ABDIL, added to the coverslips, and incubated at 37°C for 30 min or at room temperature for 1 hour. .. Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling].

Immunofluorescence:

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells
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). .. Cell viability assay Cell viability and cytotoxicity were examined by the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) and cell counting.

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Paragraph title: Interphase immunofluorescence and FISH ... Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling].

Immunoprecipitation:

Article Title: Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells
Article Snippet: Paragraph title: Protein extraction, immunoprecipitation and immunoblotting ... Immunoblotting was carried out with the following primary rabbit polyclonal antibodies: 1∶4000 anti-panZscan4 (Merck Millipore, AB4340); 1∶2000 LSD-1 (Abcam, ab37165); 1∶5000 GFP (MBL, 598), or mouse monoclonal antibodies at 1∶2000 anti-CtBP2 (BD Transduction Laboratories, 612044), 1∶5000 anti-V5 epitope (Abcam, ab27671), 1∶1000 γ-H2AX (Merck Millipore clone JBW301) or 1∶20000 anti-GAPDH (Ambion, AM4300).

Protein Extraction:

Article Title: Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells
Article Snippet: Paragraph title: Protein extraction, immunoprecipitation and immunoblotting ... Immunoblotting was carried out with the following primary rabbit polyclonal antibodies: 1∶4000 anti-panZscan4 (Merck Millipore, AB4340); 1∶2000 LSD-1 (Abcam, ab37165); 1∶5000 GFP (MBL, 598), or mouse monoclonal antibodies at 1∶2000 anti-CtBP2 (BD Transduction Laboratories, 612044), 1∶5000 anti-V5 epitope (Abcam, ab27671), 1∶1000 γ-H2AX (Merck Millipore clone JBW301) or 1∶20000 anti-GAPDH (Ambion, AM4300).

Microscopy:

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Staining was visualized on a Zeiss Axio Imager M1 microscope, with a Plan Apochromat 63× oil objective (numerical aperture, 1.4), and an AxioCam MR digital camera (Carl Zeiss). .. Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling].

BIA-KA:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cell suspension was sonicated and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 10 min, the supernatants were collected and protein concentrations were quantified using the bicinchoninic acid (BCA) method (23227, Pierce Biotechnology, Inc., Waltham, MA, USA). .. 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).

Western Blot:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: Paragraph title: 4.6. Western Blots ... 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).

Lysis:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: The cells were trypsinized and lysed in 100 µL of ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.5% SDS, 1 mM Na3 VO4 , 5 mM NaF, and 1 mM phenylmethylsulfonyl fluoride). .. 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).

Article Title: Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells
Article Snippet: To generate total cell extracts RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl pH8, 1% (v/v) NP40, 0.5%(w/v) Na Deoxycolate, 0.1% (w/v) SDS, 25 U/ml Benzonase, 1 mM sodium vanadate, 1 mM sodium molybdate, 10 mM sodium fluoride, 40 µg/ml PMSF, 0.7 µg/ml Pepstatin , 10 µg/ml Aprotinin, 10 µg/ml Leupeptin, 10 µg/ml Soyabean trypsin inhibitor) was used. .. Immunoblotting was carried out with the following primary rabbit polyclonal antibodies: 1∶4000 anti-panZscan4 (Merck Millipore, AB4340); 1∶2000 LSD-1 (Abcam, ab37165); 1∶5000 GFP (MBL, 598), or mouse monoclonal antibodies at 1∶2000 anti-CtBP2 (BD Transduction Laboratories, 612044), 1∶5000 anti-V5 epitope (Abcam, ab27671), 1∶1000 γ-H2AX (Merck Millipore clone JBW301) or 1∶20000 anti-GAPDH (Ambion, AM4300).

Software:

Article Title: Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells
Article Snippet: Immunoblotting was carried out with the following primary rabbit polyclonal antibodies: 1∶4000 anti-panZscan4 (Merck Millipore, AB4340); 1∶2000 LSD-1 (Abcam, ab37165); 1∶5000 GFP (MBL, 598), or mouse monoclonal antibodies at 1∶2000 anti-CtBP2 (BD Transduction Laboratories, 612044), 1∶5000 anti-V5 epitope (Abcam, ab27671), 1∶1000 γ-H2AX (Merck Millipore clone JBW301) or 1∶20000 anti-GAPDH (Ambion, AM4300). .. Protein relative quantification was carried out using ImageQuant RT-ECL imager and analysed using ImageQuant TL software (GE Healthcare).

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling]. .. ZEN microscopy images (.czi) were processed into extended projections of z-stacks using ZEN desk 2011 software (Carl Zeiss) and imported into CellProfiler v2.1.1 ( ) for analysis.

Fluorescence In Situ Hybridization:

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Paragraph title: Interphase immunofluorescence and FISH ... Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling].

SDS Page:

Article Title: Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells
Article Snippet: For immunoblotting, 20 µg of each protein sample or the entire immunoprecipitate was separated by SDS-PAGE and transferred to nitrocellulose as previously described . .. Immunoblotting was carried out with the following primary rabbit polyclonal antibodies: 1∶4000 anti-panZscan4 (Merck Millipore, AB4340); 1∶2000 LSD-1 (Abcam, ab37165); 1∶5000 GFP (MBL, 598), or mouse monoclonal antibodies at 1∶2000 anti-CtBP2 (BD Transduction Laboratories, 612044), 1∶5000 anti-V5 epitope (Abcam, ab27671), 1∶1000 γ-H2AX (Merck Millipore clone JBW301) or 1∶20000 anti-GAPDH (Ambion, AM4300).

Plasmid Preparation:

Article Title: Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe
Article Snippet: Plasmids and regents Cdc5L insert was subcloned into pcDNA3.0-Flag vector. .. Mouse anti-Cyclin B1 (1 : 1000) and anti-Actin (1 : 2000) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal antibody against SPF27 (1 : 1000) and anti-Prp19 (1 : 1000) were from Abcam (Cambridge, UK); mouse anti-Tubulin (1:5,000), rabbit anti-Bub1were from Sigma (St. Louis, MA, USA); mouse anti-Cdc5L (1 : 1000) was from BD Biosciences (San Jose, CA, USA); rabbit anti-PARP, anti-cleaved-Caspase3 were from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-PLRG1 (1 : 1000), rabbit anti-BubR1 were from Bethyl Laboratories Inc. (Montgomery, AL, USA); rabbit anti-phospho-histone H3 (Ser 10; 1 : 5000) and anti-γ H2AX (1 : 100) were from Merck-Millipore (Boston, MA, USA); human anti-ACA (1 : 100) was from antibodies (Antibodies Inc., Davis, CA, USA); human anti-DYNC1H1 was from Proteintech (Chicago, IL, USA; 1 : 100).

Staining:

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells
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). .. Cell viability assay Cell viability and cytotoxicity were examined by the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) and cell counting.

Article Title: Telomerase promotes formation of a telomere protective complex in cancer cells
Article Snippet: Staining was visualized on a Zeiss Axio Imager M1 microscope, with a Plan Apochromat 63× oil objective (numerical aperture, 1.4), and an AxioCam MR digital camera (Carl Zeiss). .. Antibodies used include anti-coilin (rabbit polyclonal, catalog no. Sc32860, 1:500; Santa Cruz), anti-TRF2 (rabbit polyclonal, catalog no. NB110-57130, 1:500; Novus), anti–γ-H2AX (catalog no. 05-636, 1:1000; Merck Millipore), anti-Hsp70 5A5 (mouse monoclonal, catalog no. ab2787, 1:200; Abcam), and anti-Myc [mouse monoclonal, catalog no. 2276S (9B11); Cell Signaling].

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  • 79
    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: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human gamma h2ax antibody/product/Merck KGaA
    Average 79 stars, based on 1 article reviews
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    Merck KGaA anti mouse γ h2ax
    Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for Fig 4 , followed by addition of 1 μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and <t>γ</t> H2AX assays (C) were performed as described in Figs 1 and 3 . * P
    Anti Mouse γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse γ h2ax/product/Merck KGaA
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mouse γ h2ax - by Bioz Stars, 2020-01
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    83
    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: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti s139 γ h2ax/product/Merck KGaA
    Average 83 stars, based on 1 article reviews
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    monoclonal anti s139 γ h2ax - by Bioz Stars, 2020-01
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    83
    Merck KGaA antibody against γ h2ax
    BO-1055 induces DDR and cell death A. Immunoblots showing DDR through the detection of the phosphorylation of ATM Ser1981(ATM-S1981p), Chk1 Ser 345 (Chk1-S345p), or Chk2 Thr 68 (Chk2-T68p), following the exposure of MCF-7 cells to 5, 10, or 20 μM of BO-1055 for 6-h. Cells treated with 0.1 mM of H 2 O 2 and 10 J/m 2 of UV for 30 min served as positive controls. B. The same experiment described in (A), cells were exposed to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. Immunohistochemical staining for the DNA damage marker <t>γ-H2AX</t> (green) and nucleus DAPI (blue) of cultured MCF-7 cells was conducted following incubation with 5 μM of MMC or BO-1055 for 24-h. D. FACS histogram analysis of DNA content. PI staining in fixed cells was performed following the exposure of cultured MCF-7 cells to the indicated doses of MMC or BO-1055 for the indicated times. E. FACS dot-blot analysis for cell death. AnnexinV/PI double staining in living cells was conducted following the exposure of cultured MCF-7 cells to 5 μM of MMC or of BO-1055 for the indicated times. The experiment of (D) and (E) were performed three times, and the quantitative results expressed as the mean ± SEM are respectively presented in Supplementary Figure S2A and S2B . The cell death, assessed in cells treated with 20 μM of MMC or of BO-1055, is presented in Supplementary Figure S2C .
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    Image Search Results


    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

    Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for Fig 4 , followed by addition of 1 μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and γ H2AX assays (C) were performed as described in Figs 1 and 3 . * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for Fig 4 , followed by addition of 1 μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and γ H2AX assays (C) were performed as described in Figs 1 and 3 . * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques:

    Glutathione depletion increases etoposide-mediated TOP2-DNA covalent complex formation and H2AX phosphorylation. (A) BSO preincubation (150 μ M, 4.5 hours) resulted in 70% reduction of total and reduced glutathione in NB4 cells. (B–D) NB4 cells were incubated in the presence or absence of SA (200 μ M) for 48 hours, BSO (150 μ M) for 4.5 hours, with both or with neither, followed by addition of 10 or 100 μ M etoposide for 1 hour. TARDIS and γ H2AX assays were performed as described in Figs 1 and 3 . Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Glutathione depletion increases etoposide-mediated TOP2-DNA covalent complex formation and H2AX phosphorylation. (A) BSO preincubation (150 μ M, 4.5 hours) resulted in 70% reduction of total and reduced glutathione in NB4 cells. (B–D) NB4 cells were incubated in the presence or absence of SA (200 μ M) for 48 hours, BSO (150 μ M) for 4.5 hours, with both or with neither, followed by addition of 10 or 100 μ M etoposide for 1 hour. TARDIS and γ H2AX assays were performed as described in Figs 1 and 3 . Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Incubation

    MPO activity results in raised levels of etoposide-induced H2AX phosphorylation. (A) NB4 cells were pretreated for 48 hours with SA (200 µ M) then incubated with etoposide (10 or 100 µ M). (B) K562 and K562 MPO cell lines were incubated with 10 and 100 µ M etoposide or dimethyl sulfoxide vehicle control. For both (A) and (B), γ H2AX was quantified by immunofluorescence. Analysis was carried out as described for TARDIS analysis in Fig. 1 . Data are expressed relative to the mean values obtained with 100 μ M etoposide in the absence of SA (A) or in wild-type parental K562 cells (B). Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: MPO activity results in raised levels of etoposide-induced H2AX phosphorylation. (A) NB4 cells were pretreated for 48 hours with SA (200 µ M) then incubated with etoposide (10 or 100 µ M). (B) K562 and K562 MPO cell lines were incubated with 10 and 100 µ M etoposide or dimethyl sulfoxide vehicle control. For both (A) and (B), γ H2AX was quantified by immunofluorescence. Analysis was carried out as described for TARDIS analysis in Fig. 1 . Data are expressed relative to the mean values obtained with 100 μ M etoposide in the absence of SA (A) or in wild-type parental K562 cells (B). Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Activity Assay, Incubation, Immunofluorescence

    MPO activity enhances mitoxantrone-induced TOP2-DNA covalent complex formation and H2AX phosphorylation. (A and B) NB4 cells were pretreated with 200 μ M SA for 48 hours followed by a 1-hour incubation with 0.5 or 1 μ M mitoxantrone, or a vehicle control. TOP2-DNA covalent complexes and γ H2AX were quantified as in Figs. 1 and 3 . Data are expressed relative to the mean values obtained with 1 μ M mitoxantrone in the absence of SA. (C) Quantification of MPO activity in K562 MPO line 2 compared with NB4 and parental K562 cells. (D) MPO expression in K562 cells results in enhanced mitoxantrone-induced TOP2-DNA protein complex formation. Data are expressed relative to the mean value obtained for parental K562 cells treated with 1 μ M mitoxantrone. Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: MPO activity enhances mitoxantrone-induced TOP2-DNA covalent complex formation and H2AX phosphorylation. (A and B) NB4 cells were pretreated with 200 μ M SA for 48 hours followed by a 1-hour incubation with 0.5 or 1 μ M mitoxantrone, or a vehicle control. TOP2-DNA covalent complexes and γ H2AX were quantified as in Figs. 1 and 3 . Data are expressed relative to the mean values obtained with 1 μ M mitoxantrone in the absence of SA. (C) Quantification of MPO activity in K562 MPO line 2 compared with NB4 and parental K562 cells. (D) MPO expression in K562 cells results in enhanced mitoxantrone-induced TOP2-DNA protein complex formation. Data are expressed relative to the mean value obtained for parental K562 cells treated with 1 μ M mitoxantrone. Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Activity Assay, Incubation, Expressing

    Direct MPO inhibition reduces the level of TOP2-DNA covalent complexes formation and H2AX phosphorylation induced by etoposide or mitoxantrone in NB4 cells. (A and B) NB4 cells were pretreated with MPO inhibitors PF-1355 (10 μ M, 4 hours) or MPOi-II (5 μ M, 4 hours) before adding 10 or 100 μ M etoposide or a vehicle control for 1 hour. (C and D ) NB4 cells were pretreated with MPO inhibitors as in (A and B) before adding 0.5 or 1 μ M mitoxantrone or a vehicle control for 1 hour. TOP2-DNA covalent complexes (A and C) and γ H2AX (B and D) were quantified as in Figs 1 and 3 . Numbers of replicates are indicated. ** P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Direct MPO inhibition reduces the level of TOP2-DNA covalent complexes formation and H2AX phosphorylation induced by etoposide or mitoxantrone in NB4 cells. (A and B) NB4 cells were pretreated with MPO inhibitors PF-1355 (10 μ M, 4 hours) or MPOi-II (5 μ M, 4 hours) before adding 10 or 100 μ M etoposide or a vehicle control for 1 hour. (C and D ) NB4 cells were pretreated with MPO inhibitors as in (A and B) before adding 0.5 or 1 μ M mitoxantrone or a vehicle control for 1 hour. TOP2-DNA covalent complexes (A and C) and γ H2AX (B and D) were quantified as in Figs 1 and 3 . Numbers of replicates are indicated. ** P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Inhibition

    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

    BO-1055 induces DDR and cell death A. Immunoblots showing DDR through the detection of the phosphorylation of ATM Ser1981(ATM-S1981p), Chk1 Ser 345 (Chk1-S345p), or Chk2 Thr 68 (Chk2-T68p), following the exposure of MCF-7 cells to 5, 10, or 20 μM of BO-1055 for 6-h. Cells treated with 0.1 mM of H 2 O 2 and 10 J/m 2 of UV for 30 min served as positive controls. B. The same experiment described in (A), cells were exposed to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. Immunohistochemical staining for the DNA damage marker γ-H2AX (green) and nucleus DAPI (blue) of cultured MCF-7 cells was conducted following incubation with 5 μM of MMC or BO-1055 for 24-h. D. FACS histogram analysis of DNA content. PI staining in fixed cells was performed following the exposure of cultured MCF-7 cells to the indicated doses of MMC or BO-1055 for the indicated times. E. FACS dot-blot analysis for cell death. AnnexinV/PI double staining in living cells was conducted following the exposure of cultured MCF-7 cells to 5 μM of MMC or of BO-1055 for the indicated times. The experiment of (D) and (E) were performed three times, and the quantitative results expressed as the mean ± SEM are respectively presented in Supplementary Figure S2A and S2B . The cell death, assessed in cells treated with 20 μM of MMC or of BO-1055, is presented in Supplementary Figure S2C .

    Journal: Oncotarget

    Article Title: Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms

    doi:

    Figure Lengend Snippet: BO-1055 induces DDR and cell death A. Immunoblots showing DDR through the detection of the phosphorylation of ATM Ser1981(ATM-S1981p), Chk1 Ser 345 (Chk1-S345p), or Chk2 Thr 68 (Chk2-T68p), following the exposure of MCF-7 cells to 5, 10, or 20 μM of BO-1055 for 6-h. Cells treated with 0.1 mM of H 2 O 2 and 10 J/m 2 of UV for 30 min served as positive controls. B. The same experiment described in (A), cells were exposed to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. Immunohistochemical staining for the DNA damage marker γ-H2AX (green) and nucleus DAPI (blue) of cultured MCF-7 cells was conducted following incubation with 5 μM of MMC or BO-1055 for 24-h. D. FACS histogram analysis of DNA content. PI staining in fixed cells was performed following the exposure of cultured MCF-7 cells to the indicated doses of MMC or BO-1055 for the indicated times. E. FACS dot-blot analysis for cell death. AnnexinV/PI double staining in living cells was conducted following the exposure of cultured MCF-7 cells to 5 μM of MMC or of BO-1055 for the indicated times. The experiment of (D) and (E) were performed three times, and the quantitative results expressed as the mean ± SEM are respectively presented in Supplementary Figure S2A and S2B . The cell death, assessed in cells treated with 20 μM of MMC or of BO-1055, is presented in Supplementary Figure S2C .

    Article Snippet: Cells on coverslips were then briefly rinsed with PBS and permeabilized with 0.5% Triton X-100 for 10 min, before being stained with a primary antibody against γ-H2AX (clone JBW301; Merck-Millipore).

    Techniques: Western Blot, Immunohistochemistry, Staining, Marker, Cell Culture, Incubation, FACS, Dot Blot, Double Staining

    MGMT-mediated repair is required to repair BO-1055-induced, but not melphalan-induced, lesions A. Immunoblot analysis showing endogenous MGMT expression in cells. B. DDR assessed by detecting the phosphorylation of Chk1 Ser 345 (Chk1-S345p), Chk2 Thr 68 (Chk2-T68p), or P53 Ser 15 (P53-S15p), following the exposure of HEK293T cells to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. DDR induced by BO-1055 in MGMT knockdown MCF-7 cells. D. Immunohistochemical staining of the DNA damage marker γ-H2AX (green) and the nucleus DAPI (blue) in MCF-7 cells cultured with siRNA knockdown of MGMT, followed treatment with or without 5 μM of BO-1055 for 24-h. E. Detection of DDR in MCF-7 cells transfected with control siRNA or siRNA knockdown of MGMT, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. F. Detection of DDR in HEK293T cells transfected with a control vector or an MGMT expression vector, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. G. In vitro clonogenic survival of MCF-7 cells with knockdown of MGMT by siRNA, in MCF-7 cells exposed to the indicated doses of melphalan for 6-h.

    Journal: Oncotarget

    Article Title: Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms

    doi:

    Figure Lengend Snippet: MGMT-mediated repair is required to repair BO-1055-induced, but not melphalan-induced, lesions A. Immunoblot analysis showing endogenous MGMT expression in cells. B. DDR assessed by detecting the phosphorylation of Chk1 Ser 345 (Chk1-S345p), Chk2 Thr 68 (Chk2-T68p), or P53 Ser 15 (P53-S15p), following the exposure of HEK293T cells to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. DDR induced by BO-1055 in MGMT knockdown MCF-7 cells. D. Immunohistochemical staining of the DNA damage marker γ-H2AX (green) and the nucleus DAPI (blue) in MCF-7 cells cultured with siRNA knockdown of MGMT, followed treatment with or without 5 μM of BO-1055 for 24-h. E. Detection of DDR in MCF-7 cells transfected with control siRNA or siRNA knockdown of MGMT, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. F. Detection of DDR in HEK293T cells transfected with a control vector or an MGMT expression vector, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. G. In vitro clonogenic survival of MCF-7 cells with knockdown of MGMT by siRNA, in MCF-7 cells exposed to the indicated doses of melphalan for 6-h.

    Article Snippet: Cells on coverslips were then briefly rinsed with PBS and permeabilized with 0.5% Triton X-100 for 10 min, before being stained with a primary antibody against γ-H2AX (clone JBW301; Merck-Millipore).

    Techniques: Expressing, Immunohistochemistry, Staining, Marker, Cell Culture, Transfection, Plasmid Preparation, In Vitro