Functional demonstration of a hypoxia inducible factor-1 (HIF-1) element in the human VEGF promoter region in Ha-ras–transformed cells (NIH3T3R). (A) Promoter deletion constructs derived from a 1,511-bp fragment of the VEGF promoter. The HIF-1–like wt and mutant polymers were generated by PCR amplification from the 1,511-bp fragment. Thin lines, 5′ sequences used (numbers refer to the distance from the start site of transcription); boxes, SP-1-binding sites, and solid circles, HIF-1–like element. (B) Transient transfections with HIF-1α (5 μg)- and HIF-1β (5 μg)-expression vectors with 5 μg of the HIF-1 polymer- and the HIF-1 mutant-VEGF reporter plasmid. NIH3T3R cells were transfected with reporter plasmid, allowed to recover for 17 hours in air, treated in air or hypoxia (0.02% O₂) for 5 hours before being assayed for luciferase activity. The relative fold induction refers to the ratio of luciferase activity measured in treated cells relative to the activity observed in the untreated controls. Values represent the means of at least three independent transfections. Error bars represent one standard deviation of the mean.