CSK+R extraction allows assessment of DSB repair through software-assisted Ku focus quantification. (A) Graph representing a dose–response analysis of the number of Ku foci 5 min after the indicated x-ray doses. U2OS cells were treated, postincubated for 5 min, preextracted with CSK+R, and processed. High-resolution pictures of >20 cells were acquired for each condition and submitted to automated focus detection by Volocity software. The slope given by linear regression of these data (R² = 0.962) equated to 24 Ku foci per Gy, which is less than the expected 30–40 DSBs per Gy per mammalian cell (Ciccia and Elledge, 2010). This apparent discrepancy might reflect some DSBs being blocked and unable to recruit Ku within the time frame of our experiments. Such blocks could represent certain DSBs possessing secondary structures or DNA base adducts, which must be removed for Ku to bind, and/or those residing within chromatin structures, which must be remodeled before Ku and other NHEJ components can gain access to the associated DNA ends. (B) Kinetic analysis of Ku IRIF numbers after 10 Gy of IR. U2OS cells were untreated (NT) or treated with 10 Gy of IR and postincubated for the indicated times. Cells were then processed and analyzed as in A. (C) Kinetic analysis of Ku IRIF numbers after 10 Gy of IR. U2OS cells were preincubated with DMSO, NU7441 (DNA-PKi), and/or KU55933 (ATMi) and then untreated (NT) or treated and analyzed as in B. (D) Impact of ATMi and/or DNA-PKi on survival after IR. U2OS cells were preincubated with DMSO, DNA-PKi, and/or ATMi and treated with the indicated IR doses. Inhibitors were washed away 18 h after treatment, and cell survival was determined by colony formation. For all graphs, each point corresponds to at least three independent experiments, vertical bars correspond to standard deviations, and asterisks indicate a significant difference to DMSO control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).