Fig. 4. Effects of isoflurane on hypoxia inducible factor (HIF)-1–responsive gene expression and HIF-1 transcriptional activity. (  A ) Hep3B cells were exposed to indicated stimuli for 6 h, and messenger RNA (mRNA) was analyzed. We assigned the value of 1 to the normalized HIF-1–responsive gene mRNA level on control (normoxic untreated cells). The levels of these genes in the rest of samples are represented as fold over control. (  B ) Hep3B cells were exposed to indicated stimuli for 16 h, and vascular endothelial growth factor (VEGF) protein was measured. (  C ) Hep3B cells were transiently transfected with reporter plasmids as described in the Materials and Methods. The ratio of firefly:  Renilla luciferase activity was determined and normalized to the values obtained from normoxic, wild-type transfected cells to obtain the relative luciferase activity (RLA). #  P < 0.05  versus control (normoxic untreated cells). *  P < 0.05  versus 1% isoflurane. All data are presented as mean ± SD from three replicate experiments. HO-1 = heme oxygenase 1; iNOS = inducible nitric oxide synthase.