Figure 6

TGF-β induces myofibroblastic differentiation of lung MSCs. In panel A, three different clonal cell lines were incubated for 6 days under serum-free conditions without or with 10 ng TGF-β/mL. A. qPCR reactions were run on the cDNA produced from RNA from the cells assessing the relative expression of myosin heavy chain 11 (MHC11), transgelin (TAGLN), or myocardin (MYOCD). Expression of all three was significantly elevated with TGF-β treatment (N = 3, p < 0.05). B. Western analyses demonstrated increases in α-actin and myosin heavy chain (MHC) synthesis. Signals were quantified by densitometry and normalized to β-actin (N = 3, p < 0.05). C. TGF-β increases cell size and α-smooth muscle actin expression. Forward scatter (top panel) and α-actin immunostaining (bottom panel) for cells serum deprived for 6 days (green) or treated with TGF-β (10 ng/ml) for 6 days (orange). D. TGF-β produces an increase in cell size, α-actin, and MHC immunostaining. Myofibroblastic differentiation was assessed by staining with AF488-conjugated anti-MHC (green) and anti α-actin-Cy3 conjugate (red). Co localization of α-actin and MHC Is shown In yellow to orange. White bars indicate 100 μm.