Fig 1: DNA-PKcs catalytic activity facilitates the initial recruitment of the DDR machinery to DSBs. (A) Recruitment of HR and NHEJ factors to the chromatin after IR is attenuated in the HCT116 DNA-PKcs KD/- cell line. HCT116 DNA-PKcs +/-, -/-, and KD/- cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, soluble nuclear and the chromatin-enriched fractions were isolated for immunoblotting to assess the recruitment of the proteins listed in the figure to the chromatin after irradiation. (B) IR-induced focus formation of MDC1 is attenuated in the HCT116 DNA-PKcs KD/- cell line. The HCT116 DNA-PKcs +/-, -/-, and KD/- cell lines were mock-treated or irradiated with a dose of 1 Gy and MDC1 foci formation was assessed 5 and 10 min later. MDC1 focus formation was examined in at least 50 cells and the number of MDC1 IR-induced foci per nucleus is shown. ****, P value < 0.0001. (C–F) Recruitment of GFP-tagged (C) NBS1, (D) EXO1, (E) XLF, and (F) XRCC4 to laser-generated DSBs is attenuated in the HCT116 DNA-PKcs KD/- cell line compared to the +/- cells. Relative fluorescent intensity of GFP-tagged NBS1, EXO1, XLF, and XRCC4 are presented as the mean ± SEM. *, P value < 0.05.