Fig. 4. Deubiquitination of CtIP by USP52 is important for DNA end resection and HR.

a HEK293T cells were transfected with WT CtIP or CtIP 2KR for 24 h before harvesting and immunoprecipitation with nickel (His) beads. Blots were detected by indicated antibodies. b Control or USP52-depleted ER-AsiSI U2OS cells were transfected with WT CtIP or CtIP 2KR for 24 h, and then cells were treated with 4-OHT to induce DSB. Cells were harvested for DNA end resection analysis measured by qPCR assay. Each bar represents SEM from three independent experiments. c–f Representative images (c, e) and quantification (d, f) of RPA2 (c, d) and Rad51 foci (e, f) in control or USP52 knockdown U2OS cells which were transfected with WT CtIP or CtIP 2KR for 24 h before treated with 2 Gy IR for another 2 h or 5 Gy IR for 5 h. Data are representative of three independent experiments. Each dot represents a single cell, and more than 200 cells were counted in each group for this experiment. Error bars represent SEM from this experiment. Scale bar, 10 μm. g Control or USP52-depleted HEK293T cells which were transfected with WT CtIP or CtIP 2KR were subjected to DR-GFP-based HR assay. Data are presented as mean values ± SEM from three independent experiments. h Control or USP52-depleted HEK293T cells were transfected with WT or 2KR mutant of CtIP for 24 h prior to be treated with 5 Gy IR. Cells were then harvested and immunoprecipitated with anti-FLAG agarose beads to detect the phosphorylation of CtIP at T847. i, j The sensitivity of control or USP52-depleted cells stably expressing WT CtIP or 2KR truncates in response to IR (i) or PARPi (j) were analyzed by colony formation assay. Error bars represent SEM from three independent experiments.