FIGURE 5.

Effects of Sirt7 are independent of proteasomal and lysosomal degradation. A, Hep3B cells were cotransfected with p2.1, pSV-RL, EV or Sirt7 expression vector, and vector encoding wild-type HIF-1α (WT) or triple-mutant HIF-1α (TM; P402A/P564A/N803A). 24 h post-transfection, cells were lysed, and luciferase activity was determined. B, Hep3B cells were cotransfected with p2.1, pSV-RL, EV or Sirt7 expression vector; and vector encoding wild-type or triple-mutant HIF-2α. 24 h post-transfection, cells were lysed, and luciferase activity was determined. C, Hep3B cells were cotransfected with either 1 μg of wild-type HIF-1α or HIF-2α vector or 100 ng of triple-mutant HIF-2α vector and either EV or Sirt7 vector. Total plasmid transfected was equalized with EV. 24 h post-transfection, cell lysates were analyzed by Western blotting (WB). D and E, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α (D) or HIF-2α (E) vector, and either EV or Sirt7 vector. 24 h post-transfection, cells were left untreated or treated with DMOG (1 mm) for an additional 24 h, after which cells were lysed, and luciferase activity was determined. F, Hep3B cells were cotransfected with HIF-1α or HIF-2α vector and either EV or Sirt7 vector. 24 h post-transfection, cells were treated with MG132 (20 μm) for an additional 8 h, after which cells were lysed, and Western blot assays were performed. G and H, Hep3B cells were cotransfected with p2.1, pSV-RL, expression vector encoding HIF-1α (G) or HIF-2α (H), and either EV or Sirt7 vector. 24 h post-transfection, cells were left untreated or treated with bafilomycin (5 nm) for an additional 24 h, after which cells were lysed, and luciferase activity was determined. I, Hep3B cells were cotransfected with HIF-1α or HIF-2α vector and either EV or Sirt7 vector. 24 h post-transfection, cells were treated with bafilomycin for an additional 8 h, after which Western blot assays were performed. Results are shown as means ± S.E. *, p < 0.01 for the indicated comparisons.