Figure 2. RNF168 promotes PALB2 and RAD51 recruitment and HR.

(A) Effect of the indicated siRNAs on PALB2 (white) IRIF formation at 6 hr after 10 Gy in mAG-geminin-expressing (green) S/G2 U2OS cells. (B) RAD51 (white) IRIF formation at 6 hr after 10 Gy in mAG-geminin-expressing (green) S/G2 U2OS cells transfected with the indicated siRNAs and in RNF168-deficient RIDDLE S/G2 cells complemented with either an empty vector or wild-type RNF168 expression vector. (C) Western blot analysis of RNF8 and RNF168 expression in U2OS cells treated with the indicated siRNAs. (D) Quantification of A. (E) Quantification of B. Indicated significance in D and E is compared to siLuc. (F) PALB2 IRIF formation (orange) in cyclin B1-positive S/G2 U2OS cells (white) transfected with the indicated siRNAs and siRNA-resistant GFP-tagged RNF168 cDNA (green). (G) Quantification of F. Indicated significance is compared to siLuc. (H) Effect of the indicated siRNAs on the survival of U2OS cells following treatment with PARP inhibitor (PARPi) KU-0058948. (I) Effect of the indicated siRNAs on HR efficiency measured using the DR-GFP reporter in human HEK293T cells. Indicated significance is compared to siLuc + I-SceI. (J) Western blot analysis of RNF168 expression in RNF168 mouse ES knock-out clones. (K) Effect of RNF168 knock-out on HR in two individual mouse ES clones with an integrated DR-GFP reporter. Indicated significance is compared to WT + I-SceI. Quantified data are represented as mean ± S.E.M. (n = 3), except in D where n = 2. Scale bar = 5 µm.

DOI: http://dx.doi.org/10.7554/eLife.20922.004

Figure 2—figure supplement 1. RNF168 promotes RAD51 recruitment and is epistatic with PALB2 in HR.

(A) BRCA1 or RAP80 accumulation in control (siLuc) cells or in cells depleted for either BRCA1 or RAP80 to validate the knock-down of these genes in Figure 2D. (B) Quantification of A. (C) RAD51 recruitment (white) in γH2AX-positive laser tracks (green) in mCherry-geminin-positive S/G2 cells (red). (D) Quantification of C. (E) Cell cycle profile of HEK293T cells containing DR-GFP transfected with the indicated siRNAs as shown in Figure 2I. (F) Effect of the indicated siRNAs on the survival of U2OS cells following treatment with 0.1 µM PARP inhibitor (PARPi) KU-0058948. Indicated significance in B, D, and F is compared to siLuc. The significance between siRNF168–1 and siPALB2/siRNF168–1 is also indicated in F. Quantified data represent the mean ± S.E.M. (n = 3). Scale bar = 5 µm.

DOI: http://dx.doi.org/10.7554/eLife.20922.005