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. 2014 May 14;42(12):7720–7733. doi: 10.1093/nar/gku421

Figure 4.

Figure 4.

RNF8 depletion causes a modest increase in HR in BRCA1 deficient cells that can be reversed by expression of RNF168 WT and N221*. (A) Depletion of RNF8 and BRCA1 via siRNA and expression of RNF8. U2OS cells treated with siRNAs targeting BRCA1 and RNF8 (siBRCA1#7, siRNF8#5), and a non-targeting siRNA (siCTRL). Subsequent to siRNA treatment, cells were transfected with an expression vector for RNF8 (siRNF8#5-resistant), RNF168, or EV. Shown are immunoblot signals from transfections for RNF8, BRCA1, RNF168, and actin. (B) Analysis of HDR induced by I-SceI for cells depleted of BRCA1 and RNF8. The U2OS DR-GFP reporter cell line was treated with the siRNAs described in A, and subsequently co-transfected with the expression vector for I-SceI. In addition, using samples treated with siRNAs targeting BRCA1 and RNF8, each of the RNF168 expression vectors described in Figure 2, as well as RNF8, were included in the I-SceI transfection. Shown are the frequencies of GFP+ cells for each reporter cell line, relative to parallel transfections with a non-targeting siRNA (siCTRL) and control EV. *P < 0.0001 (n = 6). (C) Analysis of HDR induced by CAS9 for cells depleted of BRCA1 and RNF8. Transfections were performed as in B, except replacing I-SceI with gRNA/CAS9 plasmids expressing gDR with CAS9-WT or CAS9-D10A, as shown in Figure 1. Frequencies of GFP+ cells were measured as in B. *P ≤ 0.034 (n = 3).