Fig. 1.

Opposite effects of HIF-1α and HIF-2α on IL-8 expression. (A) Cells were transduced for 48 h with 10, 50, and 100 MOI of AdHIF-1α and AdHIF-2α as well as 100 MOI of AdGFP as a control. Nontransduced cells were used as an additional control. Overexpression of HIF-1α and HIF-2α was confirmed by Western blotting; representative immunoblots are shown. (B) Cells were cotransfected with plasmid containing the full-length promoter of the IL-8 gene driving luciferase expression (500 ng) and plasmid containing the LacZ gene (100 ng) as an internal control and after 24 h were transduced with AdHIF-1α, AdHIF-2α, and AdGFP for the next 48 h. Next, the activity of luciferase was measured. IL-8 promoter activity was potently induced in response to HIF-2α overexpression. (C, E) Real-time PCR was performed to examine the IL-8 mRNA level after transduction with HIF-1α or HIF-2α adenoviral vectors. (D, F) ELISA was performed to assess the IL-8 protein level after 48 h transduction with adenoviral vectors. In contrast to AdHIF-1α, transduction with AdHIF-2α increased IL-8 expression at the level of both mRNA and protein. 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.