Fig. 3

DNA-PKcs suppresses mutation induction. (A) Western blot analysis showing DNA-PKcs knockdown in A549 cells by specific siRNA. The level of KU80 and GAPDH is used as loading control. Non-cropped images are provided in Figure S4A. (B) Fold increase in MF of mock transfected and DNA-PKcs depleted A549 cells, 3 days after transfection with the indicated combinations of gRNA expression vectors. Data represent the mean and SD from 3 experiments. Dots indicate the raw values of each repeat. (C) Western blot analysis showing DNA-PKcs and ATM levels in parental A549 and A549-PRKDC^−/− cells. b-Actin is used as a loading control. Non-cropped western blot images are provided in Figure S4B. (D) Western blot analysis following phosphorylation of KAP1 at Serine-824 (pKAP1-S824), as a marker for efficient activation of the IR-induced DNA damage response. The levels of KAP1 and b-Actin are used as loading controls. Non-cropped western blot images are provided in Figure S4C. (E) Fold increase in MF of parental A549 and A549-PRKDC^−/− cells, after transfection with the indicated combinations of gRNAs. Cells are plated in 6TG-selective media 3 days after transfection. Data represent the mean and SD from 3 individual experiments. Dots represent the raw values generated for each repeat. (F) Fold increase in MF of untreated A549 cells and A549 cells treated with DNA-PKcs inhibitor (NU7441, DNA-PKcsi). Data represent the mean and SD from at least two experiments. Dots represent the raw values generated for each repeat. (G) Relative change of MF between untreated and DNA-PKcsi treated A549 cells, calculated from the data shown in Fig. 3F. P-values for all panels are shown in Table S1.