Fig. 4

Interactions of MRE11:p.K464R with RAD50/RPS3. (A) Schematic diagram of IP-MS detection. (B) The directly-interacting proteins of MRE11 were screened in the String database according to the IP-MS data. (C-D) SKOV3- MRE11^WT/MRE11^K464R cells were treated with or without Olaparib for 24 h. The cells were then lysed and immunoprecipitation with MRE11 antibody (C) and the relative gray scale density of RAD50(up) and RPS3 (down) to MRE11 are presented in D.(E-F) SKOV3- MRE11^WT/MRE11^K464R cells were treated with Olaparib or DMSO. The cells were then lysed and immunoprecipitation with RPS3 antibody (E) and the relative gray scale density of RAD50 (up) and MRE11 (down) to MRE11 are presented in F. (G-H) Detection of MRE11-RAD50 (G) and MRE11-RPS3(H) interaction was carried out by PLA labeling in SKOV3 MRE11^WT/MRE11^K464R cells treated with or without Olaparib for 24 h. Representative images are shown. Scale bars, 5 μm. The scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells from three independent experiments. Data are mean ± SEM. (I-J) ER-AsiSI Hela cells were transfected with empty vector or MRE11^WT or MRE11^K464R, and then treated with 4-OHT to induce DSBs. RAD50 (I) and RPS3 (J) accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. 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