Activation of epithelial HIF-1 induces a DNA damage/replication stress checkpoint. (A) Western analysis of p21, total and phosphorylated p53S20, and phosphorylated γ-H2AX reveals induction of cell-cycle inhibitors consistent with replication stress beginning at peak endothelial proliferation (Figure 4). (B-C) Representative thin ear tissue immunofluorescent images of p21 (B) and γ-H2AX (C). White arrowheads point to p21 and γ-H2AX–positive endothelial cells. (D) Quantification of p21 and γ-H2AX expression determined by vessel counting of MECA32/p21 and MECA32/γ-H2AX double immunofluorescence tissue sections ex vivo. TetON-HIF-1 data at each DOX day were compared with TetON-HIF-1 day 0 data, using unpaired Student t test: *P < .05; **P < .01; ***P < .001. Bar represents 50 μm.
