Figure 3
From: The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage
UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E,F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G,H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) were transfected with the indicated siRNAs and subjected to clonogenic survival assay after IR (Mean ± SEM, n = 3). *p < 0.05, ***p < 0.001, N. S.; not significant. Full-length blots are presented in Supplementary Fig. S10.
