Fig. 3: ATR-mediated DDR factors recruitment in actively replicating cells is ANP32E and R-loops dependent.

a Representative p(S33)RPA32 immunofluorescence images in EdU+ cells (S-phase), untreated (left) or treated with VE822 (0.25 µM, 6 h, right). Merge shows DAPI (blue), EdU (green), and pRPA32 (red); EdU channel excluded in zoom-ins for clarity. Scale bar = 10 µm. b Quantification of pRPA32 foci/nucleus in EdU+ cells ± VE822 treatment. Unpaired two-tailed t-test p values are shown. c Representative 53BP1 immunofluorescence in EdU+ cells, untreated or VE822-treated as in (a). Merged DAPI, EdU, and 53BP1 channels shown; EdU excluded in zoom-ins. Scale bar = 10 µm. d Quantification of 53BP1 foci/nucleus in EdU+ cells ± VE822. Unpaired two-tailed t-test p values reported. e Barplots of % EdU+ cells with ≥ 1 foci for pRPA32 (left) or 53BP1 (right) under untreated conditions. Mean ± s.d. from four biological replicates; unpaired two-tailed t-test p values shown. f Barplot of % cells with pan-nuclear pRPA32 staining after VE822 (0.25 µM, 6 h) treatment. Mean ± s.d. from four replicates; p values from unpaired two-tailed t-test. g Representative images of ongoing (top) and multiorigin (bottom) replication forks. Scale bar = 10 µm. Schematic of CldU–IdU sequential labeling is shown below. h Tukey boxplots of replication fork speed (top) and inter-origin distance in multiorigin fibers (bottom) from a representative experiment. Total fibers: tIMEC = 339, tIMEC-A = 335, tIMEC-A-H1 = 248; multiorigin fibers: tIMEC = 90, tIMEC-A = 68, tIMEC-A-H1 = 43. Unpaired two-tailed t-test p values reported.