Fig. 3: Accumulation of R-loops in hypoxia (<0.1% O2).
From: Hypoxia-induced SETX links replication stress with the unfolded protein response
a A549 cells were exposed to 21% O2 or <0.1% O2 (shown in green) for 12 h, fixed and stained with the S9.6 antibody and DAPI. Where indicated, coverslips were treated with RNase H prior to staining. Data from three independent experiments (n = 3) where each dot represents a cell and a minimum of 100 cells were imaged per treatment. One-way ANOVA Tukey’s multiple comparisons test was used, ****p < 0.0001. b Representative images from a (scale bar: 5 μm). A dashed white line indicates a nuclear outline based on a DAPI stain that was used to measure the nuclear S9.6 intensity. c A549 cells were transfected with RNase HID210NV5 and exposed to hypoxia (<0.1% O2), CPT (10 µM, 20 min), or DRB (100 µM, 1 h). Staining for V5 was then carried out and the nuclear intensity determined. The two-tailed Student’s t-test was used: ****p < 0.0001. d Representative images from part c are shown (scale bar: 30 μm). V5 staining is green, DAPI to show the nucleus is blue. e HCT116 cells were transfected with mock or RNase H1 plasmid and exposed to 21% O2 or <0.1% O2 for 6 h. RNase H1 overexpression was confirmed by western blot analysis. Changes in chromatin modifications H3K9me2, H3K9me3, H3-S10, H3K4me2, H3K36me3, and H3K9Ac are shown. Total H3 was included as a loading control. f HCT116 cells were treated as in part e (<0.1% O2 for 6 h). The levels of RPA-S4/8 and Chk1-S317 are shown. RPA, Chk1, and β-actin controls were included. g HCT116 cells transfected with mock or RNase H1 plasmid and exposed to 21% O2 (Norm) or <0.1% O2 (Hyp) for 6 h were stained for RPA and the % of cells with >5 RPA foci determined. Two-tailed Student’s t-test was used: p = 0.0142. h Western blotting was carried out on cells treated as in part g. ATM activation was detected using KAP1-S824, p53-S15, and γH2AX. Total KAP1, p53, H3 and β-actin control westerns were included. a–h Data from three independent experiments (n = 3), mean ± standard error of the mean (SEM) are displayed unless otherwise indicated.
