Fig. 5

Effects of MRE11_K464R mutation on NHEJ pathway. (A) Expression of NHEJ key proteins in SKOV3 MRE11^WT /MRE11^K464R cells after treated with Olaparib for 48 h, detected by Western Blot with indicated antibodies. (B) MRE11^WT or MRE11^K464R virus were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (C) ER-AsiSI Hela cells were infected with MRE11^WT or MRE11^K464R virus, and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. (D) A2780 MRE11^WT /MRE11^K464R cells after treated with Olaparib for 48 h. The chromatin fractions and the soluble nuclear fractions were analyzed with indicated antibodies by Western Blot. (E-F) A2780 MRE11^WT /MRE11^K464R cells were treated with or with Olaparib for 48 h, and co-stained with Ku70 (green) and γH2AX (red) antibodies. E The representative images of immunofluorescence are presented, F quantification of Ku70 and γH2AX co-localization ratio per cell. Each group represents at least 100 cells counted. Scale bar, 10 μm. (G) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (H) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into ER-AsiSI Hela cells and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. (I) SKOV3 (left) and A2780 (right) MRE11^K464R cells were treated for 96 h with indicated doses of Olaparib or SCR7 (up) / AZD7648 (down) alone or combined for 96 h and viability assessed. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test