Journal: Translational Oncology

Article Title: Clinicopathological and functional evaluation of replication protein A in epithelial ovarian cancers: A target validation study

doi: 10.1016/j.tranon.2026.102709

Figure Lengend Snippet: RPA1 depletion and cisplatin sensitivity. A. Representative western blot of RPA1 knock down (KD) using siRNA in PEO4 cells. B. Clonogenic assay of cisplatin sensitivity in control PEO4 and RPA1_KD_PEO4 cells. PEO4 plating efficiency at the highest Cisplatin dose was 18 %. C. Immunofluorescent analysis of 53BP1 and γH2AX in control PEO4 and PEO4_RPA1_KD cells untreated (UT) and treated with cisplatin (5 μM) for 24 hours. D. Quantification of 53BP1 nuclear fluorescence in UT and cisplatin (5 μM) treated control PEO4 and PEO4 _RPA1_KD cells. E. Quantification of γH2AX nuclear fluorescence in UT and cisplatin (5 μM) treated control PEO4 and PEO4 _RPA1_KD cells. F. Cell cycle analysis of UT and cisplatin (5 μM) treated control PEO4 and PEO4_RPA1_KD cells G. Annexin V analysis of UT and cisplatin (5 μM) treated PEO4 and PEO4_RPA1_KD cells. H. Representative western blot of RPA1 knock down (KD) using siRNA in A2780cis cells. I. Clonogenic assay of cisplatin sensitivity in control A2780cis and RPA1_KD_ A2780cis cells. A2780cis plating efficiency at the highest Cisplatin dose was 32 %. J. Quantification of γH2AX nuclear fluorescence in UT and cisplatin (5 μM) treated control PEO4 and PEO4 _RPA1_KD cells. K. Cell cycle analysis of UT and cisplatin (5 μM) treated control A2780cis and A2780cis_RPA1_KD treated cells. L. Annexin V analysis of UT and cisplatin (5 μM) treated control A2780cis and A2780cis_RPA1_KD treated cells. UT = untreated cells; T = cisplatin treated cells. The experiment was performed for samples from three independent experiments (n=3) and the error bar represent the standard deviation (SD). ** p<0.01.

Article Snippet: Briefly, the cells were fixed with 4 % paraformaldehyde (8187,085,000, Sigma Aldrich, UK) for 30 min, permeabilised with 300 μL per well of 0.1 % Triton (85,111, Thermo Fisher Scientific, UK) for 30 min, incubated with 3 % BSA (A7906, Sigma, UK) for 1 h, incubated with 300 μL of diluted primary antibody as follows; 53BP1 [clone:4937S, Cell Signalling, 1:200, overnight at 4°C], γH2AX [clone: JBW301, Sigma, 1:500, overnight at 4°C], Alexa-fluor 488 goat anti-Mouse secondary antibody [clone: A-11,029, 1:200 Thermo Fisher, 1 h at room temperature] and Goat anti-rabbit secondary antibody, TRITC [clone: T-2769, 1:500 Thermo Fisher, 1 h at room temperature].

Techniques: Western Blot, Knockdown, Clonogenic Assay, Control, Fluorescence, Cell Cycle Assay, Standard Deviation

Journal: Translational Oncology

Article Title: Clinicopathological and functional evaluation of replication protein A in epithelial ovarian cancers: A target validation study

doi: 10.1016/j.tranon.2026.102709

Figure Lengend Snippet: RPA1 depletion and PARP inhibitor sensitivity in PEO4 cells. A. Clonogenic assay of talazoparib sensitivity in control PEO4 and RPA1_KD_PEO4 cells. PEO4 plating efficiency at the highest dose of talazoparib was 10 %. B. Immunofluorescent analysis of 53BP1 and γH2AX in control PEO4 and PEO4_RPA1_KD cells untreated (UT) and treated with talazoparib (800 nM) for 24 hours. C. Quantification of 53BP1 nuclear fluorescence in UT and talazoparib (800 nM) treated control PEO4 and PEO4 _RPA1_KD cells. D. Quantification of γH2AX nuclear fluorescence in UT and talazoparib (800 nM) treated control PEO4 and PEO4 _RPA1_KD cells. E. Cell cycle analysis in UT and talazoparib (800 nM) treated control PEO4 and PEO4_RPA1_KD cells F. Annexin V analysis of UT and talazoparib (800 nM) treated PEO4 and PEO4_RPA1_KD cells. G. Clonogenic assay of olaparib sensitivity in control PEO4 and RPA1_KD_ PEO4 cells. PEO4 plating efficiency at the highest dose of olaparib was 13 %. H. Quantification of γH2AX nuclear fluorescence in UT and Olaparib (6 μM) treated control PEO4 and PEO4 _RPA1_KD cells. I. Cell cycle analysis in UT and Olaparib (6 μM) treated control PEO4 and PEO4_RPA1_KD treated cells. J. Annexin V analysis of UT and Olaparib (6 μM) treated control PEO4 and PEO4_RPA1_KD treated cells. UT = untreated cells; T = talazoparib or olaparib treated cells. The experiment was performed for samples from three independent experiments (n=3) and the error bar represent the standard deviation (SD). ** p<0.01.

Article Snippet: Briefly, the cells were fixed with 4 % paraformaldehyde (8187,085,000, Sigma Aldrich, UK) for 30 min, permeabilised with 300 μL per well of 0.1 % Triton (85,111, Thermo Fisher Scientific, UK) for 30 min, incubated with 3 % BSA (A7906, Sigma, UK) for 1 h, incubated with 300 μL of diluted primary antibody as follows; 53BP1 [clone:4937S, Cell Signalling, 1:200, overnight at 4°C], γH2AX [clone: JBW301, Sigma, 1:500, overnight at 4°C], Alexa-fluor 488 goat anti-Mouse secondary antibody [clone: A-11,029, 1:200 Thermo Fisher, 1 h at room temperature] and Goat anti-rabbit secondary antibody, TRITC [clone: T-2769, 1:500 Thermo Fisher, 1 h at room temperature].

Techniques: Clonogenic Assay, Control, Fluorescence, Cell Cycle Assay, Standard Deviation