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. 2012 Oct 10;287(48):40618–40628. doi: 10.1074/jbc.M112.410951

FIGURE 1.

FIGURE 1.

RNF168 inhibits HDR and SSA, and depletion of RNF168 can suppress the defects in these HR events caused by BRCA1 silencing. A, shown are three reporters with an I-SceI recognition site, along with each respective DSB repair event that restores GFP+: HDR for DR-GFP, SSA for SA-GFP, and Alt-EJ for EJ2-GFP. B, depletion of RNF168 causes an increase in HDR and SSA, but not Alt-EJ. U2OS reporter cell lines were transfected with siRNF168 (pool of four siRNA sequences) prior to co-transfection of siRNF168 and an expression vector for I-SceI. Shown are the frequencies of GFP+ cells for each reporter cell line, relative to parallel transfections with a nontargeting siRNA (siCTRL). *, p < 0.0001 (n ≥ 3). Also shown are immunoblot signals for RNF168 and actin for siRNF168- and siCTRL-treated samples. C, depletion of RNF168 suppresses the HDR and SSA defects caused by BRCA1 silencing. U2OS reporter cell lines were treated with siCTRL or siBRCA1, along with (+) or without (−) siRNF168. For all (−) siRNF168 treatments, siCTRL was added to ensure equivalent concentrations of total siRNA. Repair was then analyzed as in B. Shown are the frequencies of GFP+ cells for the HDR, SSA, and Alt-EJ reporter cell lines for each siRNA treatment, relative to siCTRL. *, p < 0.0001 for HDR and SSA, p = 0.0025 for Alt-EJ, versus the (−) parallel siRNF168 condition (n ≥ 3).