FIH is involved in bortezomib-induced HIF-1α repression. (A) Bortezomib inhibited the hypoxic activation of HIF-1α. Gal4-CAD plasmid was cotransfected with Gal4-luc reporter plasmid into Hep3B cells. After incubation under normoxic or hypoxic conditions for 16 hours with bortezomib, luciferase activities were measured. The results shown are presented as relative values versus the normoxic control, and are plotted as means plus or minus SEs of 8 experiments. *P < .05 versus the hypoxic control. The reporter system is illustrated in the top panel. (B) Asn803 is required for bortezomib-induced CAD repression. Plasmid for Gal4-CAD mutant, in which Asn803 was substituted with Ala (N803A), was cotransfected with Gal4-luc reporter plasmid into Hep3B cells, and luciferase activities were measured. Results are presented as relative values versus normoxic controls in panel A, and are plotted as means plus or minus SEs of 8 experiments. The reporter system is illustrated in the top panel. (C) Verification of FIH expression and knock-down. pcDNA (0.2 μg), HA-tagged FIH (pFIH) plasmid (0.2 μg) or 20 nM control RNA (siCon), or 20 nM FIH siRNA (siFIH) was transfected into HEK293 cells. After 48 hours, FIH and β-tubulin levels in cell lysates were measured using Western blotting. (D) The effect of bortezomib on HIF-1α activity depended on FIH expression. Indicated plasmids and siRNAs were cotransfected with Gal4-CAD and Gal4-luc reporter plasmids into Hep3B cells. After incubation under normoxic or hypoxic conditions for 16 hours with bortezomib, luciferase activities were measured. □, normoxia; ▩, hypoxia; ■, hypoxia plus bortezomib. (E) EPO and VEGF expressions reduced by bortezomib were rescued by inhibiting FIH. Hep3B cells were subjected to normoxia (N) or hypoxia for 16 hours, and then cotreated with bortezomib and FIH siRNA. EPO, VEGF, and β-actin mRNAs were isolated and analyzed by semiquantitative RT-PCR.