Fig 2: 53BP1-RIF1 loss enhances resection of DSBs but is not enough to drive HR in G1 cellsAsynchronous A549 cells were treated with the indicated siRNA for 48h and irradiated with 2Gy. Cells were then fixed and immunostained for BRCA1, CtIP, and RPA after 2h or for RAD51 after 4h post irradiation. Nuclei were counterstained with CenpF to distinguish G1 and S/G2 cells. Shown are the percentages of G1 (CenpF-) cells with more than three BRCA1 A., CtIP B., RPA C., and RAD51 D. foci.

Fig 3: PTEN depletion decreases RAD51 loading. (A) RAD51 foci (green) at 3 h and 24 h after 2 Gy in CENPF-positive S/G2-phase Du145 cells transfected by either scrambled siRNA (siCTR) or siRNA targeting PTEN (siPTEN). (B) Quantitation of A. (C) RPA foci (red) at 2 h after 2 Gy in CENPF-positive S/G2-phase DU145 cells transfected by either scrambled siRNA (siCTR) or siRNA targeting PTEN (siPTEN). (D) Quantitation of the experiments performed in C. For IF experiments, at least 50 cells were analyzed. Shown are the means ± SEM from at least three different experiments. ns: not statistically significant.

Fig 4: PARP1-dependent end joining repairs the resected DSBs in 53BP1- or RIF1-depleted G1 cellsA. HeLa cells harboring pEJ were treated with the indicated siRNAs for 24h and PARP1 was then inhibited by adding 1µM olaparib 2h before transfection with I-SceI-expressing vector. The percentages of GFP+ cells were measured after 48h as indication for end joining efficiency. PARP1-EJ was measured as the percent of suppressed end joining after PARP1 inhibition. B. Representative micrographs for ?H2AX foci at 8h post 2Gy in A549 cells after depletion of either 53BP1 or RIF1 proteins in the presence or absence of olaparib. Nuclei were counterstained with CenpF to determine G1 (CenpF-) cells. C. Quantitation of ?H2AX foci number at the indicated time points in CenpF- G1 cells. D. Left panel: Western blot showing an efficient siRNA-mediated knockdown of the indicated individual or combined proteins. Right panel: Asynchronous A549 cells were treated with colcemid (0.2µg/ml) for 1h pre-2Gy and the number of ?H2AX foci was monitored in CenpF- G1 cells after the indicated treatment conditions. At least 50 nuclei were counted. (**) indicates P < 0.001. In all cases, the number of foci measured in non-irradiated cells was subtracted (relative). Shown are the mean ±SEM for three independent experiments. CTR: control, Olap: olaparib, sc: scrambled RNA, si53: si53BP1, and siR: siRIF1.