Fig 2: USP34 promotes RNF168 protein stability. (A) USP34-depleted or control cells were irradiated (10 Gy) or left untreated. Whole-cell extracts were prepared 4 h post-IR, and western blotting experiments were performed using indicated antibodies; (B) Relative expression of RNF168 (upper panel) and RNF8 (lower panel) normalized to Actin in (A) is shown. (C) 293 T cells were transfected with Streptavidin-binding peptide-Flag (SFB)-tagged RNF8, RNF168 or empty vector. Twenty-four hours post-transfection, cells were lysed in NETN, cleared by centrifugation, and streptavidin beads were added to immunoprecipitate protein complexes. (D) Amylose beads conjugated with bacterially expressed and purified MBP-RNF8 or MBP-RNF168 were incubated with 293 T cell lysate in NETN buffer for 4 h with gentle agitation at 4°C. Thereafter protein complexes were washed twice with NETN, separated by SDS–PAGE and analyzed by immunoblotting for USP34.

Fig 3: USP34 is a DNA damage-responsive protein. (A) HeLa cells transfected with USP34-targeting siRNAs (siUSP34-1 and siUSP34-2) or control siRNAs (siCTR) were seeded onto coverglasses before 2 Gy IR treatment. Cells were fixed after recovery and immunostained with anti-MDC1 antibodies to evaluate DNA repair kinetics. Western blotting experiments showed depletion of endogenous USP34 in cells treated with USP34-specific siRNAs. KAP1 expression was used as loading control. Experiments were repeated twice; (B) siRNA-treated cells were irradiated (10 Gy) and were subsequently lysed at indicated time points to evaluate level of phosphorylated KAP1 (KAP1-S824P); (C) HeLa cells transfected with indicated siRNAs were seeded onto 60 mm2 dishes, irradiated and allowed to recover for 14 days before the number of colonies were counted. Results represent data from two independent experiments each performed in triplicates; (D) HeLa cells were either untreated or treated with 10 Gy IR. Cells were processed for immunofluorescence experiments using indicated antibodies, and the percentage of USP34 foci-positive cells (of a total of 100 nuclei) before and after IR is plotted.

Fig 4: USP34 promotes DNA break-associated histone ubiquitylation. (A and B) HeLa cells were transfected with two independent USP34-targeting or control siRNAs. Cells were irradiated (3 Gy), allowed to recover for 1 h before processing for immunostaining experiments to examine focal accumulation of ubiquitylated H2A (uH2A) and conjugated ubiquitin (FK2). Percentage of uH2A and FK2 foci-positive cells (of a total of 100 nuclei) is plotted (B). (C and D) Ubiquitylation of γH2AX was examined 4 h after IR treatment (10 Gy). HeLa cells subjected to IR were lysed, sonicated and γH2AX was immunoprecipitated under denaturing condition. Western blotting experiments were performed using indicated antibodies.

Fig 5: USP34 facilitates assembly of DNA damage repair proteins. (A–C) USP34-depleted (siUSP34-1 and siUSP34-2) or control cells (siCTR) were irradiated (3 Gy), allowed to recover for 1 h before processing for immunostaining experiments were performed to evaluate focal accumulation of USP34 (A), 53BP1, RAD18 and BRCA1 (C). Percentage of cells with >10 foci (of a total of 100 nuclei) were scored as positive and quantification data is plotted (B).