Inhibition of DNA-PKcs autophosphorylation does not result in ssDNA formation. (A) The catalytic activity of DNA-PKcs was inhibited by 50 μM NU7026 or 50 μM wortmannin in M059K cells and V3+wild type DNA-PKcs cells. At 4 h after irradiation the DNA was extracted either with the warm or the cold protocol and the number of DNA fragments was detected by PFGE. The number of DNA fragments detected at 0 h by the cold protocol was set to 100%. The error bars represent the maximum deviation of two experiments (M059K+NU7026), the standard error of the mean of three experiments (M059K+Wortmannin), and the maximum deviation of duplicates from one experiment (V3+wt DNA-PKcs). (B) BrdU corresponding to ssDNA was detected 4 h after irradiation in M059K cells treated with 50 μM NU7026 and cells were scored as in Figure 3. Irradiated M059K cells without NU7026 are plotted for comparison. At least 100 cells in three experiments were scored. (C) V3 cells expressing DNA-PKcs with six autophosphorylation mutated sites (ABCDE) were treated as in (A) after irradiation. Error bars represent the SD of at least three experiments.