Journal: British Journal of Cancer

Article Title: Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes

doi: 10.1038/bjc.2017.286

Figure Lengend Snippet: Analysis of pH2AX and RAD51 foci formation. ( A ) Inmunofluorescence analysis of pH2AX induction after gamma-ray irradiation (10 Gy) was assessed in CL-V4B RAD51C WT, CL-V4B RAD51C.p.Arg312Trp, CL-V4B EV and V79B EV cells. ( B ) Percentage of cells with pH2AX foci is shown. ( C ) RAD51 foci formation was evaluated in V79B EV, CL-V4B EV, CL-V4B RAD51C WT and CL-V4B RAD51C.p.Arg312Trp cells after gamma-ray irradiation (10 Gy). ( D ) Percentage of cells with at least one RAD51 foci is presented. Non-irradiated cells (–) were included as controls. Representative images (40 ×) of each experiment are exposed. * P <0.05; ** P <0.01; *** P <0.001. Values correspond to the mean±s.e.m. of three independent experiments. A full colour version of this figure is available at the British Journal of Cancer journal online.

Article Snippet: Then, non-irradiate (control) and irradiated plates were fixed with 4% paraformaldehyde (Electron Microscopy Science, Hatfield, PA, USA) for 15 min at room temperature (r.t.) and permeabilised with 0.5% Triton X-100 for 20 min. After 30 min in blocking buffer (20% fetal bovine serum in PBS-Tween 0.1%) cells were incubated at 4 °C with mouse anti-pH2AX (Millipore) at 1 : 5000 dilution or rabbit anti-RAD51 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 100 dilution for 1 h at r.t.

Techniques: Irradiation

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR and cell cycle markers in matched normal, premalignant, and malignant human gastric specimens (A) Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers- 53BP1, H2AX, pH2AX, p53 in red; Cell cycle regulators- p16, p21 in purple. (B) Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM = Premalignant gastric lesion, M = Malignant gastric lesion. (C) Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image. Scale bar: 100 μM.

Article Snippet: After primary antibody incubation: rabbit anti-pTIF1β/pKAP1 (Ser824) (1:1000, CST # 4127S), rabbit anti-TIF1β/KAP1 (1:1000, CST # 4124S), rabbit anti-H2AX (1:1000, CST #7631), mouse anti-pH2AX (1:1000, CST #80312), rabbit anti-pDNA-PKcs (1:1000, CST, #12311S), rabbit anti-αTubulin (1:5000, CST, #2125), rabbit anti-p21 (1:500, CST, #2947S), rabbit anti-RPA32 (1:1000, Bethyl, A300-244A) and rabbit anti-pRPA32 (1:1000, Bethyl, A300-246A) membranes were washed 3 times with TBST and incubated with fluorophore-conjugated secondary antibodies diluted (1:10,000) in blocking buffer at room temperature for 1 hour.

Techniques: Staining

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR markers linked with dsDNA breaks and cell cycle regulation markers in the carcinogen-induced mouse model of gastric premalignancy (A) Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (gray), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. (B) Representative IHC images of staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5). Scale bar: 100 μM.

Article Snippet: After primary antibody incubation: rabbit anti-pTIF1β/pKAP1 (Ser824) (1:1000, CST # 4127S), rabbit anti-TIF1β/KAP1 (1:1000, CST # 4124S), rabbit anti-H2AX (1:1000, CST #7631), mouse anti-pH2AX (1:1000, CST #80312), rabbit anti-pDNA-PKcs (1:1000, CST, #12311S), rabbit anti-αTubulin (1:5000, CST, #2125), rabbit anti-p21 (1:500, CST, #2947S), rabbit anti-RPA32 (1:1000, Bethyl, A300-244A) and rabbit anti-pRPA32 (1:1000, Bethyl, A300-246A) membranes were washed 3 times with TBST and incubated with fluorophore-conjugated secondary antibodies diluted (1:10,000) in blocking buffer at room temperature for 1 hour.

Techniques: Staining, Marker

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score and CIN25 score (DNA damage checkpoint expression) for the CCLE gastric cancer cell lines. R squared value and p value calculated by simple linear correlation analysis. (B) H2AX expression (log2(TPM+1)) between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in the BROAD institute PRISM repurposing drug screen dataset; difference between the H2AX expression is represented as the mean ± S.D.; p value calculated by unpaired t test. (C) Ploidy abnormalities scores and p53 status for the five gastric cancer cell lines. (D) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in untreated five gastric cancer cell lines and RPE1 cells (non-neoplastic cell line) representing intrinsic replication stress/double-stranded DNA breaks (DSBs); nucleus stained with DAPI (blue); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines expressed as mean fluorescence intensity per cell (MFI) ± S.D from the following number of cells- RPE1-718, AGS- 2080, HGC27, 1031, NUGC3- 2119, GSU- 2092, KE39- 577. (F) Quantification of pH2AX staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (red). Data expressed as mean fluorescence intensity (MFI) of pH2AX signal per cell ± S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (G) Quantification of pKAP1 staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (green). Data expressed as mean fluorescence intensity of pKAP1 signal per cell ±S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (H) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in prexasertib (20nM) treated cell lines with intrinsic “low-replication stress”-RPE1, AGS, and intrinsic “high-replication stress”- GSU, KE39; Scale bar: 50μm.

Article Snippet: After primary antibody incubation: rabbit anti-pTIF1β/pKAP1 (Ser824) (1:1000, CST # 4127S), rabbit anti-TIF1β/KAP1 (1:1000, CST # 4124S), rabbit anti-H2AX (1:1000, CST #7631), mouse anti-pH2AX (1:1000, CST #80312), rabbit anti-pDNA-PKcs (1:1000, CST, #12311S), rabbit anti-αTubulin (1:5000, CST, #2125), rabbit anti-p21 (1:500, CST, #2947S), rabbit anti-RPA32 (1:1000, Bethyl, A300-244A) and rabbit anti-pRPA32 (1:1000, Bethyl, A300-246A) membranes were washed 3 times with TBST and incubated with fluorophore-conjugated secondary antibodies diluted (1:10,000) in blocking buffer at room temperature for 1 hour.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet:

Article Snippet: After primary antibody incubation: rabbit anti-pTIF1β/pKAP1 (Ser824) (1:1000, CST # 4127S), rabbit anti-TIF1β/KAP1 (1:1000, CST # 4124S), rabbit anti-H2AX (1:1000, CST #7631), mouse anti-pH2AX (1:1000, CST #80312), rabbit anti-pDNA-PKcs (1:1000, CST, #12311S), rabbit anti-αTubulin (1:5000, CST, #2125), rabbit anti-p21 (1:500, CST, #2947S), rabbit anti-RPA32 (1:1000, Bethyl, A300-244A) and rabbit anti-pRPA32 (1:1000, Bethyl, A300-246A) membranes were washed 3 times with TBST and incubated with fluorophore-conjugated secondary antibodies diluted (1:10,000) in blocking buffer at room temperature for 1 hour.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR and cell cycle markers in matched normal, premalignant, and malignant human gastric specimens (A) Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers- 53BP1, H2AX, pH2AX, p53 in red; Cell cycle regulators- p16, p21 in purple. (B) Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM = Premalignant gastric lesion, M = Malignant gastric lesion. (C) Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image. Scale bar: 100 μM.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Staining

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR markers linked with dsDNA breaks and cell cycle regulation markers in the carcinogen-induced mouse model of gastric premalignancy (A) Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (gray), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. (B) Representative IHC images of staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5). Scale bar: 100 μM.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Staining, Marker

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score and CIN25 score (DNA damage checkpoint expression) for the CCLE gastric cancer cell lines. R squared value and p value calculated by simple linear correlation analysis. (B) H2AX expression (log2(TPM+1)) between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in the BROAD institute PRISM repurposing drug screen dataset; difference between the H2AX expression is represented as the mean ± S.D.; p value calculated by unpaired t test. (C) Ploidy abnormalities scores and p53 status for the five gastric cancer cell lines. (D) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in untreated five gastric cancer cell lines and RPE1 cells (non-neoplastic cell line) representing intrinsic replication stress/double-stranded DNA breaks (DSBs); nucleus stained with DAPI (blue); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines expressed as mean fluorescence intensity per cell (MFI) ± S.D from the following number of cells- RPE1-718, AGS- 2080, HGC27, 1031, NUGC3- 2119, GSU- 2092, KE39- 577. (F) Quantification of pH2AX staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (red). Data expressed as mean fluorescence intensity (MFI) of pH2AX signal per cell ± S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (G) Quantification of pKAP1 staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (green). Data expressed as mean fluorescence intensity of pKAP1 signal per cell ±S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (H) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in prexasertib (20nM) treated cell lines with intrinsic “low-replication stress”-RPE1, AGS, and intrinsic “high-replication stress”- GSU, KE39; Scale bar: 50μm.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet:

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Software