Fig. 3: H2AX depletion restores replication fork protection.

a 53BP1 Ionizing Radiation-Induced Foci (IRIF) analysis in KB1P-G3 cells 4 h after 10 Gy exposure. Plotted values show the median of 53BP1 IRIF/cell from at least 600 cells (n = 2 independent experiments). P-values were calculated with one-way Anova test. Source data are provided as a Source Data file. b RAD51 IRIF analysis in KB1P-G3 cells treated as in (a). The presented data are the mean ± SD (n = 2 and n = 3 independent experiments for 53bp1-depleted and the other cell lines, respectively). Source data are provided as a Source Data file. c DNA fiber analysis in KB2P3.4 cells treated according to the depicted scheme. Hydroxyurea (HU) was used at 8 mM for 6 h. Plotted values show the median of individual IdU/CldU ratios from at least 200 fibers (n = 3 independent experiments). P-values were calculated with one-way Anova test and adjusted for multiple comparisons. Source data are provided as a Source Data file. d DNA fiber analysis in the indicated KB1P-G3 cells treated as in (c). Plotted values show the median of individual IdU/CldU ratios from at least 300 fibers (n = 3 independent experiments). P-values were calculated with two-way Anova test and adjusted for multiple comparisons. Source data are provided as a Source Data file. e Electron Microscopy (EM) analysis of reversed fork intermediates following HU treatment as in (c). The electron micrograph represents a reversed replication fork. P parental strand, D daughter strand, R regressed arm. The presented data are the mean ± SD (n = 3 independent experiments). P-values were calculated with the unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file. f EM analysis showing the median number of forks with detectable ssDNA at the junction in cells treated as in (e). P-value was calculated with the unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file. g CtIP SIRF in KB2P3.4-derived cells treated as in (c). Plotted values show the median of CtIP SIRF foci/cell from at least 140 cells (n = 3 independent experiments). P-values were calculated with one-way Anova test. Source data are provided as a Source Data file. h KB2P3.4 and KB1P-G3-derived cells were pretreated for 24 h with the indicated CtIP peptides prior to analog in vivo labeling according to the depicted scheme. HU was used at 8 mM and the CtIP peptides were used at 10 µM. Plotted values show the median of individual IdU/CldU ratios from at least 250 fibers (n = 2 independent experiments). P-values were calculated with one-way Anova test and adjusted for multiple comparisons. Source data are provided as a Source Data file.