Figure 1. MCM8-9 is required for RPA binding to DSB sites.

(a) Immunoblots of lysates from U2OS cells transfected with indicated siRNAs, 4 h after addition of cisplatin. (b) Decrease of RPA70 foci on depletion of MCM8 or MCM9. RPA70 immunofluorescence images shown at left and cells having over 20 foci of RPA70 were counted as positive cells on the right. Scale bar, 10 μm. ***P<0.005; Student's t-test. (c) ChIP assay in HeLa DR13-9 cells. Fold signal of RPA70 at I-SceI cut site relative to control site 2 kb upstream of cut site in cell treated with indicated siRNAs. *P<0.05; Student's t-test. (d) Immunoblot of U2OS cells stably transfected with empty vector (EV) or plasmid expressing siMCM8 resistant, Flag-MCM8r, 48 h after transfection of siRNAs. (e) Restoration of RPA foci formation by siMCM8-resistant Flag-MCM8. Quantification of RPA70 or γH2AX foci-positive cells as in Fig. 1b. ***P<0.005; Student's t-test. All error bars represent s.d. of the mean from triplicates. DAPI, 4′,6′-diamidino-2-phenylindole.