FIGURE 6.

Knockdown of Sirt7 expression increases HIF-1α and HIF-2α protein levels and HIF transcriptional activity. A, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α expression vector; and either empty shRNA vector (−) or vector encoding one of five shRNA sequences targeting Sirt7 mRNA (shSirt7; labeled A–E). 48 h post-transfection, cell lysates were subjected to Western blot assay. B, Hep3B cells were cotransfected with FLAG-HIF-1α vector and either empty shRNA vector or vector encoding shRNA sequences targeting Sirt7, followed by Western blotting (WB). C, Hep3B cells were treated as described for A except that HIF-2α expression vector was used instead of HIF-1α. D, Hep3B cells were cotransfected with HIF-2α vector and either empty shRNA vector or vector encoding shRNA sequences targeting Sirt7, followed by Western blotting. E and F, Hep3B (E) and HeLa (F) cells were cotransfected with p2.1, pSV-RL, and either empty shRNA vector or vector encoding shRNA sequences targeting Sirt7. 24 h post-transfection, cells were exposed to 1% O₂ for an additional 24 h and then lysed, and luciferase activity was determined. Results are shown as means ± S.E. #, p < 0.05; *, p < 0.01 versus HIF-1α, HIF-2α, or 1% O₂ alone.