Annexin A2 is up-regulated during hypoxia through Hif-1α–mediated signaling. (A) RT-PCR determination of the fold change in Anxa2 and S100a10 mRNA levels in P12-P18 retinas from Anxa2^+/+ mice after treatment with oxygen. GAPDH mRNA served as a loading control. (B) Representative immunoblot of Anxa2^+/+ retinal A2 and p11 at P12, P14, P16, and P18 after treatment with oxygen (O₂) or room air (RA). Actin was used as loading control. (C) Representative images of retinal sections from P17 Anxa2^+/+ mice either maintained in RA or treated with O₂ and stained with anti-A2 (red), isolectin B4 (green), and DAPI (blue). GCL indicates ganglion cell layer; INL, inner nuclei layer. Scale bars, 75 μm (magnification ×200). (D) Immunoblot of HIF-1α in P12, P14, P16, and P18 retinas from Anxa2^+/+ mice maintained in RA or treated with O₂. Actin served as the loading control. (E) Immunoblot and RT-PCR analyses of HIF-1α, A2, and p11 in HUVECs after treatment with either 0.5% O₂ or 200μM CoCl₂ for 16 hours. Actin served as the loading control. (F) Immunoblot and RT-PCR analyses of A2 and p11 in Hif1a^+/+ and Hif1a^−/− mouse embryonic fibroblasts after treatment with either 0.5% O₂ or 200μM CoCl₂ for 16 hours. Actin served as the loading control. (G) EMSA showing interaction between the HIF-1 complex and ANXA2 or VEGFA promoter probes. C indicates control; WT, ANXA2 or VEFGA probe containing wild-type HRE; MUT, ANXA2 or VEGFA probe containing mutant HRE; FP, free unbound probe. (H) ChIP assay showing interaction between HIF-1α and the ANXA2 promoter. Preimmune IgG and anti-RNA polymerase II served as negative and positive controls, respectively. (I) Luciferase assay showing activation of ANXA2 and VEGFA promoters by HIF-1 complex in HEK 293 cells (n = 3; *P < .05, **P < .01, and ***P < .001). The data are representative of 3 (A, C, D, G, H, and I) and 6 (B, E, and F) separate experiments.