Oxygen-dependent effects of bortezomib and FIH on the transcriptional activity of HIF-1. Gal4-CAD/Gal4-luciferase reporter plasmids (A,C) or EPO enhancer-luciferase reporter plasmid (B,D) were cotransfected with cytomegalovirus-β-gal plasmid into HEK293 (A,B) or MCF7 (C,D) cells. Gal4-CAD(N803A)/Gal4-luciferase reporter plasmids were cotransfected with the β-gal plasmid into HEK293 and MCF7 cells (E). HA-tagged FIH (pFIH) plasmid, FIH siRNA (siFIH), or CITED2 siRNA (siCITED2) was also cotransfected into these cells. After being stabilized for 48 hours, transfected cells were incubated under normoxic (N) or hypoxic (1% or 0.5% O₂) conditions for 16 hours with 1 or 10 nM bortezomib (4 hours prior to hypoxia), and then luciferase activities were measured. The results shown are presented as relative values versus the hypoxic control, and are plotted as means (± SD) of 8 or more experiments. *P < .01 versus the hypoxic control; #P < .01 versus the 1 nM bortezomib group; ##P < .01 versus the 10 nM bortezomib group; NS, not significant versus the hypoxic control.