HIF-2α increases IL-8 expression independent of Nrf2. (A–D) Cells were transduced for 48 h with AdHIF-1α, AdHIF-2α, or AdGFP as a control. Real-time PCR was performed to examine the mRNA levels of Nrf2, HO-1, NQO1, and Bach1. Western blotting was done using antibodies against Nrf2, HO-1, and Bach1; representative immunoblots are shown. Note a decrease in Nrf2 and its target genes’ expression and a concomitant increase in the expression of Bach1, the Nrf2 repressor. (E) Real-time PCR after 48 h transfection with siRNA targeted against Nrf2 mRNA or scrambled siRNA. Nrf2 siRNA abolished the Nrf2 mRNA expression. (F) ELISA was performed to assess the protein level of IL-8 after 24 h transfection with siRNA against Nrf2 mRNA or scrambled siRNA followed by 48 h transduction with AdHIF-2α or AdGFP. Silencing of Nrf2 did not influence HIF-2α-induced IL-8 production compared to cells transduced with the AdGFP vector, in which Nrf2 was also silenced. Each bar represents the mean ± SD of two to six independent experiments performed in duplicate. *p < 0.05, comparing AdGFP vs AdHIF-1α or AdHIF-2α.