Fig. 6. Incorporation of biotinylated rED-A into the cellular FN network. 3D shadow projections, views from basal side (A,B). Single optical sections (C,D). Bar, 10 µm. (A) Fibroblasts treated with TGFß for 4 days. ED-A FN (green) displays a parallel organisation of thick fibers. (B) TGFß-treated fibroblasts in the presence of rED-A (red). Yellow staining shows zones of colocalisation. The organisation of the FN network is less regular upon rED-A treatment compared to TGFß treated fibroblasts (A). Biotinylated rED-A displays a preferential incorporation into the FN network near the periphery of the cell. Note the thickening of FN fibers mainly at the places of rED-A incorporation. (C,D) Desoxycholate (DOC)-resistant matrix deposited by TGFß-treated cells (C) or by TGFß-treated cells incubated with biotinylated rED-A (D): rED-A (red) and ED-A FN (green). Note a focal incorporation of rED-A into the fibers of DOC-insoluble FN matrix (D). As for B, yellow staining represents zones of colocalisation. (E) DOC-soluble (lanes 1 and 2) and -insoluble (lanes 3 and 4) fractions were prepared from fibroblasts cultured for 4 days in 5 ng/ml of TGFß in the absence (lanes 1 and 3) or in the presence of 100 µg/ml of biotinylated rED-A (lanes 2 and 4). Corresponding amounts of proteins were resolved on 6% gels (FN), 8% gels (vimentin) and 12% gels (rED-A) and probed with a rabbit polyclonal antibody to FN or streptavidin, and with a monoclonal antibody to vimentin. rED-A is incorporated in the DOC-insoluble matrix but does not reduce the level of DOC insoluble FN or of vimentin.
