Inhibition of DNA repair by knockdown of PHAX. (A) U2OS cells were transfected with the indicated siRNAs, followed by UV-irradiation (20 J/m2). After 6-h incubation, phase-contrast images were obtained. (B,C) U2OS cells were transfected with the indicated siRNAs, followed by ADR (B) or CPT (C) at the indicated concentrations. After 24-h incubation, cell viabilities were determined by alamarBlue assay. (D) U2OS cells were treated as in A. The indicated protein levels were determined by western blotting analysis. (E–H) U2OS cells were cotransfected with the indicated siRNAs and each reporter plasmid for DNA repair assay (pDRGFP for HDR [E,F] or pEJ2GFP for NHEJ [G,H]). Fluorescence microscopic images were obtained, and the numbers of GFP-positive cells were counted. As a positive control, the cells were treated with PARP inhibitor (PARPi) at 100 nM for 48 h. Data are the means ± S.D. (n = 4). (*) P < 0.01, (**) P < 0.001.
