USP7-disrupted cells show reduced activation of HRR. (A) Normal healthy donor PBMCs and 3 isogenic CLL cell lines (2 p53 wild type [wt] and 1 p53 defective [def]) expressing the indicated shRNAs show diminished expression and thus destabilization of RAD18 protein after treatment with HBX19818. β-actin was the loading control (Con). (B) HeLa cells transfected with USP7 or Con siRNAs and empty (Vec) or siRNA-resistant Flag-USP7 (R-USP7)–expressing plasmid vectors demonstrate rescue of RAD18 stability in a complementation assay. The reduction of RAD18 levels in the presence of USP7 siRNA alone is prevented by the expression of R-USP7, which replaced the endogenous USP7 depleted by USP7 siRNA. (C,D) Representative immunofluorescence labeling of DNA damage markers γH2AX and RAD51 (green) with DAPI-counterstained nuclei (blue); (E-H) quantification of RAD51 foci formation from 24-hour time-course studies of 5-Gy IR-induced damage (n = 3). In MEC1 cells (C,G), normal healthy donor cells (E), and primary CLL cells (F), HBX19818 induced inhibition of USP7 1 hour before irradiation abrogated the formation of IR-induced RAD51 foci. (H) IR-induced RAD51 foci formation was reduced in HeLa cells after USP7 depletion by USP7 siRNA. (I) Analysis of HRR using the DR-GFP reporter assay in U2OS cells showed that HRR is compromised by USP7 depletion. CtIP depletion was the positive Con for HRR-defective cells. Original magnification ×60 (C-D). Data were compared using a 2-tailed Student t test, and statistical significance denoted by: *P ≤ .05, **P ≤ .01. Ub, ubiquitylated.