Silencing of SLX4–XPF suppresses accumulation of DDR factors at lacO, and the subsequent accumulations of ATR and FANCD2 are interdependent. (A) U2OS 40–2-6 ER-LacI cells were transfected with control (siGL2) or SLX4 (siSLX4-3)-targeting siRNAs for 48 h. The cells were further treated with 1 µM 4-OH-tamoxifen (4-OHT) for 2 h, then subjected to colocalization analysis by double immunostaining. Similar experiments using other siRNAs are shown in Fig. S4 B. (B–D) U2OS 40–2-6 ER-LacI cells were transfected with control (mixture of siGFP and siLuci) siRNAs or siRNAs targeting XPF (siXPF-1 and siXPF-2), MUS81 (siMUS81-2), or FANCD2 (siFANCD2-2) for 48 h. (B and C) Following siRNA treatment, cells were treated with 1 µM 4-OHT for 2 h and subjected to colocalization analysis. (D) At 42 h after siRNA transfection, cells were treated with 1 µM 4-OHT for 6 h and then analyzed. Similar experiments using other siRNAs are shown in Fig. S4, F and H. (E) U2OS 40–2-6 ER-LacI cells were treated with 10 µM ATR inhibitor (VE-821) or vehicle only (DMSO) for 6 h, with 1 µM 4-OHT for the last 2 h, followed by colocalization analysis. For A and B, the means ± SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test). For C–E, the values were calculated from the sum scores of two independent experiments. **, P < 0.01; ***, P < 0.001; n.s., not significant (χ² test). Individual data points from the two independent experiments are also depicted.