Figure 1

TGF-β1-mediated activation of PSCs leads to downregulation of miR-29 and increases ECM protein expression.

Mouse PSCs (mPSCs) and human PSCs (hPSCs) were serum starved for 24 hours and stimulated with 10 ng/ml of TGF-β1 for 24 hours. Total RNA or protein were isolated for qPCR and western blot analysis respectively. (a) qPCR analysis of miR-29 family members in nascent and TGF-β1 stimulated mPSCs and hPSCs. (b) Activation of pSMAD2/3 in TGF-β1 stimulated mPSCs. Serum starved mPSCs were treated with TGF-β1 for 6 hours and subjected to western blot analysis for pSMAD2/3 and total SMAD2/3 expression. β-actin and GAPDH were used as loading controls. (c) qPCR and immunofluorescence analysis of PSC activation marker, alpha smooth muscle actin (α-SMA) in TGF-β1 stimulated mPSCs. Representative images are shown, α-SMA (red) and nuclear stain, DAPI (blue). (d) qPCR analysis of TGF-β1 responsive transcriptional gene target, connective tissue growth factor (CTGF) in nascent and TGF-β1 stimulated mPSCs. (e) Western blot analysis of extracellular matrix proteins: collagen 1alpha1 (collagen 1a1), collagen 3alpha1 (collagen 3a1), laminin gamma-1 (laminin) and fibronectin in nascent and TGF-β1 activated mPSCs. α-tubulin, or GAPDH, or β-actin were used as loading controls. Relative quantification of band intensities, normalized to loading controls, are shown below respective blots. Experiments were repeated 3-4 times and representative data are shown. Data is presented as the mean + standard error of the mean (SEM); n = 3; p-values determined by t-test, *p < 0.05, **p < 0.01.