PAI-1 loss results in dysregulated miR-21 expression, which promotes AKT-mediated cell proliferation of skeletal muscle fibroblasts. (A) uPA activity (chromogenic assay), active TGF-β1 (ELISA), active TGF-β1 protein, and miR-21 expression (quantitative PCR) were analyzed in muscle fibroblasts from PAI-1^+/+ and PAI-1^−/− mice, either untreated or treated with recombinant PAI-1 (rPAI-1) and amiloride or transfected with siRNA for uPA or Scramble control, as indicated (*, P < 0.05; means ± SEM; n = 4 for each group). (B) PAI-1^+/+ and PAI-1^−/− muscle fibroblasts, transfected with Ant-miR-21 or Scramble oligomiR, were stimulated or not with 10 ng/ml TGF-β1, and the number of cells was counted at the indicated time points (means ± SEM; n = 4 for each group). (C) PAI-1^+/+ and PAI-1^−/− muscle fibroblasts, stimulated as in B, were analyzed for Coll I and TIMP-1 expression after TGF-β1 treatment (*, P < 0.05; means ± SEM; n = 4 for each group). (D) Extracts of PAI-1^+/+ and PAI-1^−/− muscle fibroblasts, stimulated or not with TGF-β1 for 24 h, were analyzed by Western blotting using antibodies for the proteins P-Smad2, total Smad2/3 protein, P-AKT, total AKT protein, PTEN, and β-actin; in lane 5, PAI-1^−/− muscle fibroblasts were incubated with 2 µM wortmannin (Wort) 30 min before the addition of TGF-β1. In lanes 6 and 7, PAI-1^−/− muscle fibroblasts were transfected with Scramble and siRNA uPA oligomiRs, respectively, before the addition of TGF-β1. (E) Western blotting analysis of muscle fibroblasts (left) or extracts of gastrocnemius muscles from 3-mo-old PAI-1^+/+ and PAI-1^−/− mdx mice (right) treated every other day for 1 mo with a scrambled oligomiR or Ant-miR-21 using antibodies for the proteins P-AKT, total AKT, PTEN, and β-actin.