Fig. 2. Loss of ATM induces unrepaired DNA damage in normoxia and after reoxygenation in human PAEC and in EC-Atm-/- mice.
a–c Commercially available healthy donor PAEC were cultured under hypoxia (0.5% O2) for 48 h followed by room air for 24 h (reoxy), or in room air for 72 h (normo). a Representative immunoblot of DNA damage markers (γH2AX and phosphorylated RPA) in human PAEC transfected with scrambled control siRNA (siC) or siRNA targeting ATM (siA). n = 3 individual experiments. b Comet assay shows DNA damage as reflected in the comet tail lengths in ATM-depleted cells in normoxia or reoxygenation. Scale bar, 20 µm. Cells n = 186, 154, 139, 124 for siC normoxia, siA normoxia, siC Reoxy, siA reoxy respectively. c Immunohistochemistry of the DNA damage markers in human PAEC transfected with Control or ATM siRNA. Scale bars, 20 μm. Cells n = 130, 133, 117, 102 for siC normoxia, siA normoxia, siC Reoxy, siA reoxy respectively. d Animal measurements - experimental design: Mice with EC-specific deletion of Atm (EC-Atm-/-) were created using the strategy described for Fig. 1, and in the “Methods”. EC-Atm-/- or control mice were exposed to hypoxia (10% oxygen) for 3 weeks followed by 4 weeks of room air (reoxy), or maintained in room air for 7 weeks (normo). Schema created with BioRender.com. e γH2AX immunofluorescence foci in PAEC in control and EC-Atm-/- mice (arrowheads) were quantified in n = 6 mice. Scale bars, 20 μm. The bottom panels show a magnified merged image of the area delineated by the dotted line. Scale bars, 5 μm. In (a and d), bars represent mean ± S.E.M. P values determined by 2-way ANOVA with Holm-Sidak posthoc test. ns, not significant. In (b and c) The Bounds of boxes show the 25th and 75th percentiles, the whisker showed to the 10th to 90th percentiles and the centre in the box shows the median. Three independent experiments was performed. P values determined by Kruskal-Wallis ANOVA test with Dunn’s test Source data are provided as a Source Data file.