Fig. 1.

Mammalian target of rapamycin complex (mTORC)1 inhibition reduces transforming growth factor (TGF)-β-induced type I collagen expression in human mesangial cells (HMC). A: HMC were treated with mTORC1-specific inhibitor (rapamycin 10 nM) or DMSO for 30 min and then with TGF-β1 (1 ng/ml) or vehicle for 24 h. Whole cell lysate was analyzed by Western blot for type-I collagen. Each experiment was performed at least 3 times, and data from a representative experiment are shown. B: HMC were transfected with the COL1A2 promoter construct and treated in triplicate with rapamycin 10 nM or DMSO for 30 min and then TGF-β1 (1 ng/ml) or vehicle for 24 h. Luciferase assay values corrected for transfection efficiency using β-galactosidase (β-gal) controls, are shown (means ± SE). C: HMC were transduced with lentiviral particles containing short-hairpin (sh)RNA to Raptor or scrambled control, and knockdown of Raptor was evaluated by Western blotting with whole cell lysate harvested after 48-h incubation with the shRNA. D: HMC were cotransfected with the COL1A2 promoter construct and shRNA for Raptor or 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). Results shown in the graphs are the combination of 3 separate experiments (*P <0.05, #P <0.05).