Figure 6.

TNS1 is required for fibrillar adhesion formation and extracellular matrix (ECM) deposition by myofibroblasts. (A) Coimmunostaining for TNS1 and β1-integrin in HLFs transfected with TNS1 siRNA or scrambled control, followed by treatment with 1 ng/ml TGF-β for 24 hours. Scale bars, 50 μm. Immunostaining for the row is indicated in the images of the left column. (B) Coimmunostaining for TNS1 (green) and fibronectin (red, extracellular fibrils) in HLFs. Image digitally zoomed for clarity. Scale bar, 25 μm. (C) Quantitation of number and length of fibrillar (activated β1-integrin–containing) adhesions in TGF-β–induced (1 ng/ml, 24 h) myofibroblasts under siRNA-mediated TNS1 knockdown or scrambled control. Mixed-effect ANOVA (* and ^#P < 0.05) was used for statistical analysis. (D) HLFs, transfected with TNS1 siRNA or scrambled control, were treated with 1 ng/ml TGF-β for 72 hours, then replated and incubated with Alexa488-labeled fibronectin (488-FN) for 90 minutes, followed by fixation and immunofluorescent microscopy. Mixed-effect ANOVA (* and ^#P < 0.05) was used for statistical analysis. (E and F) HLFs were transfected with TNS1 siRNA or scrambled control, treated with 1 ng/ml TGF-β for 72 hours, followed by deoxycholate (DOC) buffer extraction to remove non–ECM-bound material. Residual ECM was then solubilized and run via PAGE followed by Western blotting and densitometry of the indicated proteins. Data show reduced DOC-insoluble fibronectin (FN) or collagen I (Col I) fractions. Mixed-effect ANOVA (*P < 0.05) was used for statistical analysis. * and # symbols designate significant difference between the indicated conditions. Data are presented as means (±SD). A.U., arbitrary units.