Fig. 5.
TGF-β induces increased HIF expression in a Smad3- and TORC1-dependent manner. A: HEK were transfected with FLAG-tagged WT Smad3 or pEXL EV for 24 h and then treated with TGF-β1 (1 ng/ml) or vehicle for 6 h. Whole cell lysate was analyzed by Western blot for HIF-1α. Experiments were repeated 3 times, and sets of results from a representative experiment are shown. B: HMC were cotransfected with the HRE-luc construct and a construct containing WT Smad3 or pcDNA3 EV and then treated with TGF-β1 (1 ng/ml) or vehicle for 24 h. Luciferase assay results, corrected for transfection efficiency using β-galactosidase controls, are shown (means ± SE). C: transformed human mesangial (tHMC) cells stably expressing scrambled shRNA or shRNA directed against Smad3 lysate were analyzed by Western blot for Smad3. D: Smad3 knockdown tHMC cells or control were transfected with the HRE-luc construct and treated in triplicate with TGF-β1 (1 ng/ml) or vehicle for 24 h. Luciferase assay values, corrected for transfection efficiency using β-galactosidase controls, are shown (means ± SE). E: HMC were cotransfected with the HRE-luc construct and construct containing WT Smad3 or pEXL EV. Cells were treated with rapamycin (10 nM) or vehicle for 30 min and then with TGF-β1 (1 ng/ml) or vehicle for 24 h. Luciferase assay values, corrected for transfection efficiency using β-galactosidase controls, are shown (means ± SE). Each luciferase experiment was performed in triplicate. Results shown for all graphs represent combination of 3 separate experiments (*P <0.05, #P <0.05).