Increased TGF-β activity regulates miR-21 expression and fibrosis in dystrophic muscle of DMD patients, mdx mice, and in injured WT muscle. (A) Representative immunostaining of muscle biopsies of DMD patients and healthy control subjects using an anti–P-Smad2 antibody. Bar, 25 µm. (B) Active TGF-β1 protein quantification by ELISA in muscle biopsies of DMD patients and healthy controls. (C) The expression levels of Coll I, TGF-β1, TIMP-1, and FN1 from the same human muscle biopsies used in B are shown. (B and C) *, P < 0.01; means ± SEM. n = 7 patients 6–12 yr of age; n = 6 healthy 9–15 yr of age. (D) Representative immunostaining of diaphragms of WT and mdx mice of 3 mo of age using anti–P-Smad2 antibody. Bar, 25 µm. (E) Active TGF-β1 protein quantification by ELISA in the diaphragm muscle of WT and mdx mice of the indicated age (*, P < 0.01; means ± SEM; n = 4 for each group). (F) Fibroblasts obtained from skeletal muscle were cultured in vitro and treated with TGF-β1 for 18 h, and the expression of miR-21 and its polyadenylated precursor (Pri-miR-21) was analyzed by quantitative PCR (*, P < 0.05; means ± SEM; n = 5 for each group). (G) Muscle fibroblasts were left untreated (NT) or were treated with the indicated combinations of TGF-β1 (24 h), Ant-miR-21, Mimic-miR-21, or Scramble oligomiR. The expression of Coll I and TIMP-1 was analyzed by quantitative PCR (*, P < 0.01; **, P < 0.001; means ± SEM; n = 4 for each group).