Fig. 2: ANP32E overexpression results in increased R-loops, TRCs, and DNA damage.

a Dot blot showing R-loop abundance in tIMEC, tIMEC-A, and tIMEC-A-H1 cells ± RNaseH1. dsDNA used as internal normalizer. Quantification from three biological replicates (mean ± s.e.m.), unpaired two-tailed t-test p values shown. b Representative PLA images showing proximity between ANP32E and R-loops. Scale bar = 10 µm. c PLA images of pS2RNApol II-PCNA proximity as TRCs marker. Scale bar = 10 µm. d Quantification of PLA foci/nucleus from (b, c): tIMEC n = 106/104, tIMEC-A n = 85/84, tIMEC-A-H1 n = 72/75. Unpaired two-tailed t-test p values are shown. e Top: FANCD2 immunofluorescence in EdU+ cells (EdU channel excluded for clarity). Bottom: quantification of FANCD2 foci/nucleus in EdU+ (green violins) and EdU– (gray violins) cells; unpaired two-tailed t-test p values shown. Scale bar = 10 µm. f Alkaline comet assay ± VE822 (1.25 µM, 24 h). Top: representative images, comet quantification (OpenComet)⁹⁴, and DNA intensity heatmaps (CometScore)⁹⁵. Bottom: violin plots of comet tail length; 150–300 cells/condition, three biological replicates. Unpaired two-tailed t-test was used for p value calculation. g Left: representative binucleated (BN) cells with micronuclei (MNi) after Cytochalasin B + Mitomycin C treatment. Right: %BN cells with MNi ± Mitomycin C (0.03 µg/ml) or VE822 (0.027, 0.08, 0.25 µM) treatment. ≥3000 cells/condition on three biological replicates. The barplot reports mean ± s.d. with two-way ANOVA adjusted p values. h Immunostaining of BN cells with MNi that are positive or negative for centromere staining (CREST) after VE822. Phalloidin marks cytoplasm; arrows indicate MNi. Scale bar  = 10 µm. i Barplot showing the percentage of BN cells with DSB-derived (acentric) MNi ± Mitomycin C or VE822 treatment. Mean ± s.e.m. from three replicates; 25–60 BN cells/condition were quantified per replicate.