Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2, Col1a1, and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, *p < 0.05 compared to serum-free control).