CSK+R extraction enables observations into mechanisms controlling repair pathway choice. (A) U2OS cells were preincubated for 1 h with DMSO or ATMi and then treated for 1 h with camptothecin (CPT) before being preextracted with CSK+R and processed for immunofluorescence. Cells in S phase were identified as showing pronounced γ-H2AX staining upon camptothecin treatment. The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Insets represent twofold zoom to highlight Ku80 microfoci (boxed regions). Bars: (white) 10 µm; (green) 1 µm. (B) U2OS cells were preincubated 1 h with DMSO, ATMi, and/or DNA-PKi before being treated with DMSO (NT, not treated) or 1 µM camptothecin for the indicated times (minutes). At the end of the treatment, cells were preextracted with CSK+R and processed for immunofluorescence. Ku foci were quantified as in Fig. 5 A in cells in S phase untreated or treated with camptothecin. NonS columns represent Ku focus numbers in non–S-phase cells treated for 1 h with CPT. Each bar corresponds to at least three independent experiments. (C) Total extracts from MEFs derived from wild-type (WT) or XRCC5^−/− mice were analyzed by immunoblotting. (D) MEF wild type or XRCC5^−/− were preincubated for 1 h with DMSO or 10 µM ATMi and then treated with the indicated doses of camptothecin. Inhibitors and camptothecin were washed away 18 h later, and cell survival was determined by colony formation. Each point corresponds to three independent experiments. Error bars correspond to standard deviations.