Figure 1.

USP4 Promotes DSB Repair

(A) USP4-targeting siRNAs depleted USP4 from U2OS cells.

(B) USP4 depletion from U2OS cells caused DSB repair defects (neutral comet assays) after phleomycin (40 μg/ml, 2 hr) treatment, measuring the effects of depleting CtIP or DNA ligase IV, as controls (mean ± SEM; n = 3). Also see Table S1 for siRNAs and Table S2 for antibodies used in this study.

(C) Treatments of U2OS cells with USP4-2 but not USP4-UTR siRNAs depleted exogenously expressed GFP-USP4 WT(L); exog, exogenous; endog, endogenous. Also, see Table S3 for plasmids used in this study.

(D) Exogenously expressed USP4 WT(L) restored DSB repair defects (neutral comet assays) observed after USP4 depletion (mean ± SEM; n = 3).

(E) USP4 depletion sensitized U2OS cells to IR (mean ± SEM; n = 3 and XRCC4 siRNA-treated cells were the positive control).

(F) Treatment of U2OS cells with USP4-UTR siRNAs depleted endogenous USP4 but not exogenously expressed GFP-USP4 WT(H) or CD. exog, exogenous; endog, endogenous (also see Figure S1C for expression levels).

(G) Expression of GFP-USP4 CD but not WT(H) caused DSB repair defects (neutral comet assays) irrespective of endogenous USP4 depletion (mean ± SEM; n = 3).

(H) Expression of GFP-USP4 CD sensitized U2OS cells to IR (mean ± SEM; n = 3).

(I) GFP-USP4 WT and CD immunoprecipitations from U2OS cell extracts retrieved FLAG-USP4 WT (^∗p < 0.05 ^∗∗p < 0.01; n.s., not significant). See also Figure S1.