FIGURE 3.

BCAS2 and PSO4 are required for the recruitment of ATRIP to DNA damage sites and the subsequent CHK1 activation and RPA2 phosphorylation. A and B, BCAS2 or PSO4 depletion impairs CHK1 activation and RPA2 phosphorylation after DNA damage. HeLa cells infected with the indicated shRNAs were either mock-treated or treated with CPT (1 μm) (A) or IR (10 gray) (B) for 1 h. Cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates a nonspecific band. sh-Con, control shRNA. C and D, BCAS2 or PSO4 depletion impairs CPT-induced phospho-RPA2 focus formation. BCAS2- or PSO4-depleted HeLa cells were treated with CPT (1 μm) for 1 h before fixing and processing for phospho-RPA2 immunofluorescence. Representative phospho-RPA2 foci are shown (C). Quantification results were the average of three independent experiments and are presented as mean ± S.E. (D). More than one hundred cells were counted in each experiment. E and F, BCAS2 or PSO4 depletion impairs CPT-induced ATRIP focus formation. BCAS2- or PSO4-depleted HeLa cells were treated with CPT (1 μm) for 1 h before fixing and processing for ATRIP immunofluorescence. Representative ATRIP foci are shown (E). Quantification results were the average of three independent experiments and are presented as mean ± S.E. (F). More than 100 cells were counted in each experiment.