Fig 2: 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 3: 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 4: ATM regulates 53BP1-RIF1-mediated end protection by phosphorylating 53BP1 in a cell cycle dependent mannerA. Representative micrographs for p53BP1 (S25/29; left panel & S1778; right panel) foci in asynchronous A549 control cells (DMSO) or after inhibition of either ATM (ATMi) or DNA-PK (DNA-PKi). Nuclei were counterstained with CenpF to distinguish G1 (CenpF-) from S/G2 (CenpF+) cells. B. Quantitation of the experiments presented in ‘A’. About 100 nuclei were counted. C. Focus intensity of pATM in G1 and S/G2 cells at the indicated time points after 2Gy. The intensities of about 2000 foci were quantified in each cell cycle phase. D.-E. Foci number of p53BP1 at D. S25/29 or E. S1778 in G1 vs S/G2 cells at the indicated time points after 2Gy. F. A549 cells were treated with the indicated siRNAs and p53BP1 (S1778) foci were enumerated at 2h post-2Gy in G1 and S/G2 phases. G. pATM focus intensity of about 2000 foci was measured at 1h post-2Gy in G1 and S/G2 A549 cells treated as in ‘F’. Data presented as the mean ±SEM of two (for p53BP1 foci) or three (for pATM focus intensity) independent experiments.