Fig. 6.
Inhibitory effect of mTORC1 inhibition on collagen expression can be reversed by HIF-1α overexpression. A and B: HMC were cotransfected with the HRE-luc construct (A) or COL1A2-luc construct (B) and a construct containing nondegradable (ND) HIF-1α or pcDNA3 EV. Cells were treated in triplicate with rapamycin (10 nM) or DMSO for 30 min followed by addition of 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). Results shown are the combination of 2 separate experiments (*P < 0.05, #P < 0.05). C and D: HMC were cotransfected with the HRE-luc construct (C) or COL1A2-luc construct (D), a construct containing nondegradable HIF-1α or pcDNA3 EV, and shRNA for Raptor or control scrambled shRNA. After 20 h the cells were treated 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; *P <0.05, #P <0.05). Each luciferase experiment was performed in triplicate. Results shown are combination of 2 separate experiments (*P < 0.05, #P < 0.05). E: HMC were treated with rapamycin (10 nM) or vehicle for 30 min, then treated with deferoxamine (DFO) 100 μM or vehicle for 30 min, followed by addition of TGF-β1 (1 ng/ml) or vehicle for 24 h. Whole cell lysate was analyzed by Western blot for collagen I. Representative set of immunoblots is shown from 3 separate experiments.