Figure 1.

Mfs activate latent TGF-β1 from self-generated ECM depots. (A–D and G) Lung Mfs were stained for LTBP-1 (green), ED-A FN (red), α-SMA (blue), and nuclei (insets) after a 2- (A), 3- (B), or 7-d (C) culture, and after extracting all cellular components with DOC at day 7 (D). (E) Expression of LTBP-1, ED-A FN, and α-SMA was further evaluated together with TGF-β RII and LAP by Western blotting; vimentin served as loading control. Whole culture extracts were compared with samples taken after both TX-100 (TX; preserves the cytoskeleton and ECM) and DOC (preserves ECM) extraction. LTBP-1 was blotted in nonreducing conditions after digesting DOC-insoluble ECM overnight with plasmin. (F) Lung Mf (2–7 d) and subcutaneous fibroblasts (7 d) were examined for total TGF-β1. Total TGF-β1 was measured by incubating TMLC with supernatants from 80°C heat-activated trypsinized cells (cellular TGF-β1), as well as from heat-activated, DOC-insoluble ECM (ECM-associated total TGF-β1). Function-blocking TGF-β1 antibody was added to test TGF-β isoform specificity (dashed lines). TGF-β1 concentration was calculated from a standard. (G) Cultures of subcutaneous fibroblasts were stained after 7 d as in A–D. (H, left) Total culture extracts were produced from lung Mfs, lung fibroblasts (lung F), subcutaneous fibroblasts (sub F), and subcutaneous Mfs (sub Mf) and blotted as in E. Gray lines indicate that intervening lanes have been spliced out. (H, right) The DOC-insoluble fraction of 7-d Mf was used as latent TGF-β1–containing ECM for co-culture of fibroblasts or Mfs with TMLC. TGF-β1 activated by cells during 24 h was measured as luminescence compared with 24-h control cultures on plastic culture dishes. Error bars represent the SD of the mean. Bars: (A–D and G) 20 μm; (insets) 200 μm.