Figure 3.

Effect of FIH-1 on HIF-1-mediated reporter gene transcription. Human 293 (A,D) or Hep3B (B,C) cells were cotransfected with pSV-Renilla, a reporter gene containing the SV40 promoter and Renilla luciferase-coding sequences, p2.1, a reporter gene containing a 68-bp hypoxia-response element upstream of the SV40-promoter and firefly luciferase-coding sequences, and the indicated amount of expression vector containing FIH-1 cDNA [inserted in either the sense or antisense (AS) orientation], HIF-1α cDNA, or empty vector (EV). For each expression vector, the amount (in nanograms) of plasmid DNA transfected is indicated. (E) Hep3B cells were co-transfected with pSV-Renilla, reporter pG5ElbLuc, containing five GAL4-binding sites upstream of an adenovirus Elb promoter and firefly luciferase coding sequences, expression vector encoding the GAL4 DNA-binding domain alone (Gal0) or fused to HIF-1α residues 531–826 (GalA), and EV or vector encoding FIH-1. In each panel, cells were exposed to 20% (open bars) or 1% (closed bars) O₂ for 16 h and the ratio of firefly:Renilla luciferase activity was determined. The results were normalized to those for cells transfected with EV and exposed to 20% O₂ (relative luciferase activity). The mean and standard deviation based on 3–9 independent transfections are shown.