Fig. 5
Loss of hCINAP leads to a defect in error-free HR pathway. a hCINAPWT and hCINAP−/− HEK293T cells were subjected to HR assays. The data are presented as the mean ± SEM of three replicates. More than 1000 cells were counted in each group, two-tailed students’ t test, ***P < 0.001. b hCINAPWT and hCINAP−/− U2OS cells treated with or without IR (10 Gy) were subjected to immunofluorescence assays. Representative immunofluorescence images are shown (left), and statistics of BRCA1 foci numbers in each group are shown (right). ****P < 0.0001. c NPM1 knockdown U2OS cells transfected with the indicated plasmids were treated with IR (10 Gy) and released for 1 h. The accumulation of BRCA1 was then assessed. Scale bar, 10 μm. d Schematic of the role of different BRCA1 complex in end resection. e–g hCINAPWT and hCINAP−/− U2OS cells were immunostained with anti-RAP80 or anti-CtIP antibodies. Representative immunofluorescence images are shown in e, and BRCA1 foci numbers in cells were counted in f and g. Scale bar, 10 μm. ****P < 0.0001. h, i Immunofluorescence h and its quantification ishowing that hCINAP deficiency blocked the RAD51 foci recruitment. Representative images and percentages of cells with > 10 RAD51 foci were counted. The U2OS cells were treated with 4 Gy IR and released 4 h before fixing. Scale bar, 10 μm. ***P < 0.001. j HEK293T cells transfected with the indicated vectors were subjected to co-IP analysis using the indicated antibodies. k Cells were exposed to 10 Gy IR and then recovered for 1 h before being subjected to immunofluorescence. Scale bar, 10 μm. Additional cell images and quantification of RAD51 foci are shown in Supplementary Fig. 12. For b, c, f, g, the results are shown as the mean ± SEM from three independent experiments. Data were analyzed using Student’s t test. More than 100 cells were counted per group. Unprocessed scans of blots are provided in Supplementary Fig. 13
