Figure 2.

mSin1 is required for mTORC regulation and fibrotic functions of mesenchymal cells. A, nonfibrotic MCs (n = 4) were transfected with scrambled or mSIN1-specific siRNA and then treated with LPA (10 μm for 6 h). Densitometry of the replicates was analyzed. Values are means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B–D, cell lysates were subjected to immunoblot analysis for phosphorylated and total forms of AKT (Ser-473, B), p70S6K1 (Thr-389, C), and 4E-BP1 (Thr-37/46, D). Densitometry of the replicates was analyzed. Values are means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, nonfibrotic MCs and fibrotic MCs (n = 10) were analyzed for mSin1 protein expression by immunoblotting, and the densitometric analysis is presented as ***, p < 0.001. F, mesenchymal cells derived from fibrotic lung allografts (fibrotic MCs, n = 6) were transfected with scrambled or mSIN1-specific siRNA, and the lysates were immunoblotted with antibodies against mSin1, collagen I, phosphorylated and total forms of AKT (Ser-473), TSC2 (Thr-1462), FOXO3a (Ser-253), p70S6K1 (Thr-389), and 4E-BP1 (Thr-37/46) using GAPDH as a loading control. G, fibrotic MCs (n = 10) were transfected with siRNA specific to LPAR1 or the scrambled control, and the lysates were immunoblotted with antibodies against LPA1, collagen I, and mSin1 as well as the phosphorylated and total forms of AKT (Ser-473), p70S6K1 (Thr-389), and 4E-BP1 (Thr-37/46) using GAPDH as a loading control. Representative blots are shown.