Figure 5. Ablation of PTIP rescues homologous recombination in BRCA1 deficient cells.

(A) WT, BRCA1^−/−, PTIP^−/− and BRCA1^−/−PTIP^−/− B cells were pulsed with CFSE and stimulated with (red) or without (green) PARPi. CFSE signal diminishes with increasing division. BRCA1^−/− cells are sensitive to PARPi (arrow indicates sluggish cells) but loss of PTIP in BRCA1-deficient cells rescues the proliferation defect. (B) Analysis of genomic instability (radial chromosomes, chromatid breaks and chromosome breaks) in metaphases from B cells treated with 1 μM PARPi. At least 50 metaphases were analyzed for each genotype. (C) B cells were stimulated for 2 days, irradiated with 10 Gy and the percentage of cells with immunofluorescent RAD51 foci were quantified (at least 400 cells counted for each genotype). Data in (B) and (C) represent mean of three experiments +/− standard deviations. **: p<0.05 (two-tailed unpaired t-test), ns: not significant. (D) High-throughput microscopy quantification of RPA foci per cell in WT and PTIP^−/− MEFs that were either untreated or treated with 30 Gy IR. Top: representative image of chromatin bound RPA in irradiated WT and PTIP^−/− cells. Bottom: quantitation of RPA foci. Bar indicates the mean number of RPA foci per cell, and the blue box designates cells with more than 15 foci, whose percentage is indicated above each box. **: p<0.001 (E) BRCA1^−/−PTIP^−/− B cells were reconstituted with PTIP^WT or PTIP^W663R retroviruses (expressing a GFP marker driven by an internal ribosome entry site) and treated with PARPi. Cells were sorted (GFP^positive =infected and GFP^negative =uninfected) and metaphases were analyzed for radial chromosomes (n=50 metaphases analyzed in each case). See also Figures S6-S9.