Fig. 1: DNA resection (ssDNA) correlates with nascent RNA synthesis.
From: CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway
a Representative immunofluorescence images of EU, BrdU, PCNA and DAPI staining in U2OS cells depleted for the indicated DDR factors, irradiated with 5 Gy and stained upon 30-minute labeling with EU and BrdU that started immediately after IR exposure. Scale bar: 10 μm. b Graph shows nucleoplasm (non-nucleolar) EU intensity in different cell cycle phases in non- and irradiated-U2OS cells. EU component was added upon DNA damage using 5 Gy in irradiated cells and labeled for 30 min in both cellular conditions. c Graph shows EU intensity in different cell cycle phases in control U2OS cells irradiated and labeled for 30 min as in (a). d Graph shows intensity of BrdU staining under non-denaturing conditions to visualize stretches of ssDNA, as a DNA resection marker in cell cycle phases of U2OS cells irradiated (5 Gy). e Bar graph represents nucleoplasm (non-nucleolar) EU intensity in cells treated by siRNAs against indicated genes in non- and irradiated cells. EU component was added upon DNA damage using 5 Gy in irradiated U2OS cells and labeled for 30 min in both cellular conditions. Mean values and ± s.e.m are represented from at least 500 cells of 3 independent experiments. Irradiated samples comparison show **p = 0.0062 (siNT vs siCtIP), **p = 0.0018 (siNT vs siBRCA1) and ***p = 0.0001 (siNT vs siRAD52) using multiple comparison with Ordinary Two-Ways ANOVA. f Nascent RNA synthesis in U2OS cells depleted for the indicated DDR proteins, labeled with EU for 30 min starting after irradiation with 5 Gy. g Quantification of BrdU staining under non-denaturing conditions to mark ssDNA as a DNA resection marker in U2OS cells depleted for CtIP, BRCA1 and RAD52, respectively, and stained after 30 min BrdU labeling started after exposure to 5 Gy. Statistical data at (b–d) and (f, g) showing mean data from 3 independent experiments. Bar plots show the median (center), 25–75 percentile (box), and 5–95 percentile (whisker) from at least 500 cells. P values were calculated using multiple comparison with Ordinary One-Way ANOVA. ***p < 0.001. Source data are provided as a Source data file.
