Figure 7. Hif-1α mediates the reduction of S100A1-3′UTR reporter gene expression.

A) EA.hy926 (upper panel) or primary HMVEC (lower panel) cells were exposed to the prolyl-hydroxylase-2 inhibitor IOX2 (10 µmol/L) for 24 h to induce Hif1-α, prior to extract preparation. A representative immunoblot is shown to verify Hif1-α induction. The experiment was done 3 times with similar results. β-actin was used to control for protein loading. B) Expression levels of MiR-138 were assessed by qPCR in extracts prepared from EA.hy926 ECs subjected to 24 h treatment with 10 µmol/L IOX2. C) EA.hy926 ECs were co-transfected with the S100A1–3′UTR luciferase reporter gene and either the antimir-138 or scramble control (Dharmacon). 24 h later cells were incubated with IOX2 to induce Hif1-α stabilization. *, P<0.02 vs untreated. For both B, C, the experiment was done 3 times, each in triplicate. D) EA.hy926 ECs were transfected with the S100A1-3′UTR reporter gene and co-transfected with siRNA against Hif1-α, or control scramble siRNA. Cells were then subjected to chemical hypoxia with 250 µmol/L CoCl₂ for 24 h before luciferase activity was assessed. *, P<0.02 vs normoxic, P<0.05 vs siRNA Hif1-α. Experiment was performed 3 times, each in triplicate.