Figure 4.

Overexpression of PAI-1 or PAI-2 leads to the upregulation of myofibroblastic markers. To evaluate whether the overexpression of PAI-1 and PAI-2 phenocopy the myofibroblastic phenotype of Cav-1-deficient fibroblasts, the expression of several myofibroblastic markers was assessed in fibroblast control or overexpressing PAI-1 or PAI-2. (A) Localization of calponin (upper parts) and vimentin (lower parts). Note that overexpression of PAI-1 or PAI-2 upregulates the levels of two myofibroblastic markers, calponin and vimentin. Nuclei were immunostained with Hoechst (blue). Images captured at original magnification of 63x. (B) Immunoblotting for vimentin and fibronectin. Note that overexpression of PAI-1 or PAI-2 upregulates vimentin and the overexpression of PAI-1 increases the expression of fibronectin. Immunoblotting with β-tubulin is shown as a control for equal loading. (C) Localization of fibronectin in fibroblast-derived extracellular matrices. Fibroblasts overexpressing PAI-1 or PAI-2 along with controls were plated onto gelatin cross-linked coverslips. Fresh medium containing ascorbic acid was added every other day for 5 d to induce extracellular matrix production. Then, the cells with their matrices were fixed and immunostained with an antibody against fibronectin. Note that PAI-1 overexpression increases fibronectin deposition, a myofibroblastic marker. Nuclei were counterstained with Hoechst (blue). Images captured at original magnification of 63x. Ctrl, fibroblasts containing empty vector alone; PAI-1, fibroblasts overexpressing PAI-1; PAI-2, fibroblasts overexpressing PAI-2.