(A) Purified MECs from WT and Rnf8^–/– littermate females at estrus phase were either left untreated (UT) or irradiated (5 Gy) and fixed at the indicated time points after IR. Cells were stained with anti-γH2ax antibody and counterstained with DAPI. (B) Quantitative analysis of γH2AX subnuclear foci in WT and Rnf8^–/– MECs from A. Cells with more than 3 foci were counted as foci-positive. Dot plots show mean ± SEM of 3 independent experiments, and more than 100 cells for each condition and genotype were counted. (C) Representative images of spontaneous γH2AX subnuclear foci in Rnf8^–/– Trp53^Δ/Δ mammary tumor cells reconstituted with RNF8 or mock (empty vector). (D) Dot plots depicting quantification of the spontaneous subnuclear foci formation for γH2AX, 53BP1, BRCA1, and RAD51 observed in Rnf8^–/– Trp53^Δ/Δ mammary tumor cells reconstituted with RNF8 or mock. Three independent experiments were performed, and more than 100 cells were counted for each condition and genotype (mean ± SEM). (E) Representative images of γH2AX, 53BP1, BRCA1, and RAD51 subnuclear foci formation 6 hours after IR (5 Gy) of the indicated mammary tumor cells. (F) Dot plots depicting quantifications of subnuclear foci formation observed in E. Cells harboring at least 10 foci were counted as foci-positive. Three independent experiments were performed, and more than 100 cells were counted for each condition and genotype (mean ± SEM). (G and H) Dot plots depicting efficiencies of HR-mediated (G) and NHEJ-mediated (H) repair of I-SceI–induced DSBs in Rnf8 Trp53^Δ/Δ mammary tumor cells reconstituted as indicated (mean ± SEM). Three independent experiments were performed. Two-sided Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 20 μm.