Recruitment of various DDR proteins to lacO arrays in U2OS 40–2-6 ER-LacI cells. (A) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT for 2 h were double immunostained with the indicated antibodies and counterstained with DAPI. ssDNA was stained with anti-BrdU antibody under nondenaturing conditions. For detection of total ssDNA, DNA was labeled for 24 h with 10 µM BrdU. For detection of nascent-strand ssDNA, newly synthesized DNA was labeled with BrdU during treatment with 4-OHT. For detection of parental-strand ssDNA, DNA was labeled with 10 µM BrdU for 20 h, followed by a chase in fresh medium without BrdU for 2 h before addition of 4-OHT. Representative images are shown, and the colocalization frequencies are shown in Fig. 1 D. Yellow arrows indicate colocalization of DDR proteins with the foci of HA-LacI, and white arrows indicate noncolocalization. Scale bars, 10 µm. (B) The antibody used in this study can detect bleomycin-induced RAD51 foci. U2OS 40–2-6 ER-LacI cells were treated with 8 µM Bleomycin for 18 h, then immunostained with anti-RAD51 antibody, followed by DAPI staining. Representative images are shown. Scale bar, 10 µm. (C) U2OS 40–2-6 ER-LacI cells treated with 1 µM 4-OHT or a control vehicle (EtOH) for 2 h were subjected to ChIP-quantitative PCR analysis using the indicated antibodies. Enrichment of the lacO sequences is shown as the percent of input DNA. Means ± SD are shown (n = 6 for control rabbit IgG; n = 7 for LacI and 53BP1). *, P < 0.05; ***, P < 0.001; n.s., not significant (two-tailed Student’s t test).