Fig. 7
The cancer-associated USP15 mutation resulted in homologous recombination (HR) defect. a A schematic representation of breast cancer-associated USP15 mutations. b USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or deletion mutant (DM) and HR efficiency were determined. Data are presented as mean±SD of three independent experiments. Student's t test, *P < 0.05, **P < 0.01. c–e USP15-knockout U2OS cells stably reconstituted with USP15 WT or DM mutants were subjected to IR (4Gy). Representative immunostaining of BARD1 (c), RPA (d), and RAD51 (e) are shown. Scale bar: 10 μm. f USP15-depleted MCF7 cells were reconstituted with USP15 WT or DM mutant. Cells’ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were performed by colony formation assay. Data are presented as mean±SEM of three independent experiments. Two-way analysis of variance (ANOVA), **p < 0.01. g The 293T cells transfected with FLAG-USP15 WT or indicated mutants were lysed and immunoprecipitated by FLAG M2 beads. Samples were analyzed by Western blot with indicated antibodies. h USP15-knockout 293T cells were reconstituted with HA-USP15 WT or DM, and treated as indicated for 24 h. Cells were lysed and ubiquitinated BARD1 were pulled down by Ni-NTA under denatured condition and blotted with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Fig. 10. NS not significant
