ECM maturation and preremodeling affects TGF-β1 activation by myofibroblasts. (A) hDMfs were grown for 2, 3, and 6 d, and lysates were used for Western blotting with vimentin as a loading control. (B) hDf- or hDMf-derived ECMs were decellularized using DOC and heat activated before total amounts of TGF-β1 were determined by luciferase reporter assay. (C) hDMfs were seeded onto different preproduced ECMs and stimulated to contract with thrombin, and active TGF-β1 was measured as a percentage of total TGF-β1. (D) hDMfs were transfected with LTBP-1–EGFP and stained for LTBP-1 and EGFP after 12 d. (E) EGFP, ED-A FN, and nuclei (blue) were stained in hDMf cultures after 2, 3, and 6 d. Bottom rows show magnified ECM regions. (F) The mean density of LTBP-1–EGFP fibrils was quantified from stained images. Bars: (D) 150 µm; (E, top) 25 µm; (E, bottom) 5 µm. Graphs show mean values and SDs from at least three independent experiments (*, P ≤ 0.05; and ***, P ≤ 0.005 using ANOVA followed by a post-hoc Tukey’s multiple comparison test). a.u., arbitrary unit.