HIF-1α repression by amphotericin B requires FIH. (A) FIH mRNA levels. FIH overexpression and suppression by siRNA were evaluated by RT-PCR method. (B-C) Augmentation of the AmB-induced CAD repression by FIH expression. The plasmid (0.2 μg) for pcDNA or HA-tagged FIH was cotransfected with Gal4-CAD and Gal4-luc reporter plasmids into Hep3B cells. After incubation under normoxic (N) or hypoxic (H) conditions for 16 hours with AmB, luciferase activities were measured. (D-E) Abolishment of the AmB-induced CAD repression by knocking down FIH. siRNA (20 nM) of control (si-con) or FIH (si-FIH) was cotransfected with Gal4-CAD and Gal4-luc reporter plasmids into Hep3B cells. After normoxic or hypoxic incubation with AmB for 16 hours, luciferase activities were measured. (F) Status of FIH activity under normoxic or hypoxic conditions. FIH siRNA (20 nM), pcDNA (0.2 μg), or pHA-FIH (0.2 μg) was cotransfected with Gal4-CAD and Gal4-luc reporter plasmids into Hep3B cells. After normoxic or hypoxic incubation for 16 hours, luciferase activities were measured. Results are quoted as relative values versus the normoxic value in pcDNA-transfected cells and are the means ± SD of 8 experiments. The averages of inhibition percent versus the hypoxic control or P values versus the pcDNA control are presented in the figures.