Fig. 4.

mTORC1 signaling is Smad-dependent. pHLFs were transfected with control siRNA or siRNA targeting Smad3 and Smad3 protein expression measured by Western blot (a). Following transfection, pHLFs were stimulated with TGF-β₁ (1 ng/ml) for 12 h and mTORC1 signaling evaluated by Western blot analysis (b). An irrelevant lane has been spliced out of the prepared images (a) and (b). Uncropped gels are shown in Supplementary Fig. 9. COL1A1 mRNA levels were assessed by real-time RT qPCR at 24 h (n = 3) (c). Relative expression was calculated using 2^−ΔCt. ΔCt was calculated from the geometric mean of two reference genes. Collagen I deposition was measured by macromolecular crowding assay at 72 h (d). Data are expressed as collagen I signal normalized to cell count (n = 4 fields of view imaged per well) calculated as a percentage of the TGF-β₁ -treated control (n = 5). Data are presented as mean ± SEM and are representative of 2 independent experiments. Differences between groups were evaluated with two-way ANOVA with Tukey multiple comparison testing, ***p < 0.001, ****p < 0.0001