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. 2012 Jan 4;287(9):6139–6149. doi: 10.1074/jbc.M111.278630

FIGURE 5.

FIGURE 5.

FHL proteins inhibit HIF-1 transcriptional activity in hepatocellular carcinoma cells. A, FHL1–3 inhibit HIF-1 activity in Hep3B cells subjected to hypoxia. Hep3B cells were co-transfected with pSV-RL; p2.1; FLAG-HIF-1α; and either EV or vector encoding FHL1, FHL2, or FHL3. 24 h post-transfection, cells were exposed to an additional 24 h under nonhypoxic or hypoxic conditions. Cells were then lysed and the ratio of firefly:Renilla activity was measured. B, FHL1–3 inhibit HIF-1α TAD function. Hep3B cells were co-transfected with pSV-RL; pG5-E1b-Luc; either Gal4-HIF-1α(531–826) or Gal4-EV; and either EV or FHL1, FHL2, or FHL3 vector. 24 h post-transfection, the cells were lysed to determine firefly:Renilla activity. C–E, FHL1–3 inhibit HIF activity at target gene promoters. Hep3B cells were co-transfected with the indicated luciferase promoter constructs and either EV, FHL1, FHL2, or FHL3 vector. 24 h post-transfection the cells were exposed to an additional 24 h under nonhypoxic or hypoxic conditions. The cells were then lysed, and luciferase activity was measured. F, FHL1–3 knockdown increases HIF-1 activity under hypoxic conditions. Hep3B cells were co-transfected with pSV-RL; p2.1; and either empty vector (shEV) or vector encoding shRNA against FHL1, FHL2, or FHL3. 24 h post-transfection the cells were exposed to an additional 24 h at 20% or 1% O2. The cells were lysed, and the ratio of firefly:Renilla activity was determined. G, FHL1–3 knockdown increases HIF-1 activity under nonhypoxic conditions. Hep3B cells were co-transfected with pSV-RL; p2.1; FLAG-HIF-1α vector or EV; and either EV or vector encoding shRNA against FHL1, FHL2, or FHL3. 48 h post-transfection the cells were lysed, and the ratio of firefly:Renilla activity was determined. H, FHL1–3 knockdown up-regulates HIF-1α TAD function. Hep3B cells were co-transfected with pSV-RL; pG5-E1b-Luc; either Gal4-HIF-1α(531–826) or Gal4-EV; and either EV or vector encoding shRNA against FHL1, FHL2, or FHL3. 24 h post-transfection the cells were exposed to 24 h of hypoxia and then lysed to determine firefly:Renilla activity. The amount of total plasmid transfected was kept constant in all experiments. The results are shown as the means ± S.D. *, p < 0.01; #, p < 0.05 compared with EV or shEV.