Figure 3.

VE-821 inhibits hypoxia-induced ATR-mediated signaling, while inducing DNA damage and affecting replication kinetics. (A) RKO cells were exposed to ⩽0.02% O₂ for the times indicated in the presence of DMSO or 1 μℳ VE-821. Western blotting was carried out as indicated. Representative blots of n=3 experiments are shown. The levels of β-actin were determined to ensure equal loading. Quantification is shown in Supplementary Figure S2. (B) RKO cells were treated with DMSO or 1 μℳ VE-821 as shown. Norm-6 h; Hyp-6 h ⩽0.02% O₂. The percentage of cells with more than six nuclear 53BP1 foci is shown. (C–F) VE-821 decreases replication fork progression and increases origin firing. RKO cells were treated with DMSO or 1 μℳ VE-821 and were treated as indicated, either in normoxia or ⩽0.02% O₂ for 6 h and subsequently reoxygenated for 1 h. DNA fibres were then produced and scored. (C, D) The percentage of different replication structures is shown. (E, F) The distribution of the fork rates for the second label (IdU) for the on-going forks (average speeds are indicated). Significance values: *P<0.01, **P<0.001, ***P<0.0001.