ECM disorganization suppressed TGF-β1 activation FAK^−/− MEFs, and wild-type MEFs were cultured for 6 d. (A) ECM production was assessed by immunofluorescence staining for FN (green) and nuclei (blue), and the mean density of FN fibrils was calculated from six images per three independent experiments. Bar, 100 µm. (B) Culture lysates were immunoblotted using vimentin and GAPDH as loading controls. (C) Cells were seeded for 1 d onto silicone substrates, and wrinkle formation as a result of substrate deformation was quantified from phase-contrast images. Bar, 25 µm. (D) After 6-d culture, MEFs were removed using DOC, and total TGF-β1 was measured after heating the remaining ECM. (E) hDMfs were seeded onto the decellularized MEF ECMs; active TGF-β1 levels were measured as a percentage of total TGF-β1 without (baseline) and with thrombin-induced contraction. (F) TMLCs alone were seeded onto decellularized ECM produced by MEF wild type (WT) and FAK^−/− and stimulated for 1 h with 0, 0.03, 0.06, 0.12, 0.25, 0.50, and 1.0 ng/ml active TGF-β1. Cells were further processed and assessed for luciferase production (luminescence). The data shown are from a single representative experiment, and the experiment was completed once with three independent measurements. (G) Decellularized MEF ECM was incubated for 1 h with magnetic beads coated with BSA or anti-LAP (aLAP) antibody before magnetic force was applied, and active TGF-β1 was measured in the supernatant. Graphs show mean values and SDs from at least three independent experiments (***, P ≤ 0.005 using ANOVA followed by a post-hoc Tukey’s multiple comparison test). a.u., arbitrary unit.