Fig. 5. USP42 is epistatic with DHX9 in the cellular survival after DSB induction and promotes resolution of DSB-induced R-loop.

a, b The interaction between USP42 and DHX9 was tested by coimmunoprecipitation with either GFP-USP42 a or GFP-DHX9 b followed by immunoblotting analysis with the indicated antibodies. Transfection with a plasmid encoding GFP was a negative control. c U2OS cells transfected with the indicated siRNAs were treated with CPT and then subjected to immunoblotting analysis with the indicated antibodies. d A DR-GFP assay was performed with the cells transfected with the indicated siRNAs (mean ± SEM, n = 3). e BRCA1 foci formation efficiency was examined with cells transfected with the indicated siRNAs. (Upper) The representative images are shown. (Lower) The population of cells with >10 BRCA1 foci was plotted (mean ± SEM, n = 3). The cells positive for CENPF staining were analysed. See Fig. S4B for the representative images of undamaged condition. f U2OS or USP42 KO cells transfected with the indicated siRNAs were subjected to a clonogenic survival assay (mean ± SEM, n = 5). g, h The indicated cells g or cells transfected with the indicated siRNAs h were treated with phleomycin (Phleo., +) and then further cultured for 2 h upon removal of phleomycin (2 h). Purified genomic DNA was subjected to slot blot analysis with an S9.6 antibody. Signal intensity was normalized to mock-treated samples (−), and then plotted as a Box and Whiskers plot (median, 5–95 percentile, n = 5 for U2OS, USP42 KO, and siBRCA1, n = 7 for siCtrl). *p < 0.05, ***p < 0.005.