Figure 6.

Activation of latent TGF-β1 by Mf contraction increases with increasing ECM stiffness. (A) Lung Mfs were cultured for 7 d on silicone substrates with increasing stiffness (Young's modulus). TMLC reporter cells were added for a 4-h direct co-culture to assess active TGF-β1 with and without promoting Mf contraction using 0.5 U/ml thrombin. (B) Total TGF-β1 content was measured in the supernatant of heat-activated DOC-insoluble ECM produced from each substrate condition; values are expressed as luminescence in the TMLC reporter system. (C) Protein expression was evaluated by Western blotting normalized for vimentin expression and by immunostaining (D–F) for α-SMA (red), F-actin (green, phalloidin), and nuclei (blue) after 7 (D and E) and 8 h (F) of culture on 5-kPa compliant (D and F) and 47-kPa stiff (E) substrates. ECM was extracted with DOC from 7-d Mf cultures on compliant (G) and stiff (H) substrates and immunostained for LTBP-1. (I) Mfs were freshly seeded onto Mf-derived, DOC-insoluble ECM and cell-activated TGF-β1 was evaluated after 8 h of direct co-culture with TMLC as luminescence. Error bars represent the SD of the mean. Bar, 100 μm.