siRNA down-regulation of WRN and DNA ligase IIIα in CML cell line. (A) Nuclear extracts were prepared at 4 different time points (0h, 24h, 48h, and 72h) after siRNA treatment of cells. Western blotting was performed using WRN antibody (90% knockdown; top panel) and DNA ligase IIIα antibody (75% knockdown; middle panel). siRNA using nontarget oligonucleotides were used as controls. Ku86 was used as loading control (bottom panel). (B) Bar graph showing the percentage of K562 cells with more than 5 γH2AX foci examined in siRNA nontarget, siRNA knockdown of WRN and DNA ligase IIIα cells with or without irradiation (IR; 2.5 Gy). Black bars indicate no IR and gray bars are with IR treatment. Two hundred nuclei were examined from each of 3 different experiments. Values significantly different are marked with an asterisk (P < .01 by Student t test). Error bars reflect the standard error of the mean. (C) Images of 3 different cells showing γH2AX foci (FITC, green signal) in K562 following siRNA nontarget (left panels), siRNA down-regulation of WRN (middle panels), and DNA ligase IIIα (right panels), with and without irradiation.