FIGURE 3.

HADD inhibits FN assembly by fibroblasts. A, mouse FN^−/− cells adherent to laminin-coated coverslips were given 20 nm FITC-FN in the absence (NA) or presence of 50 or 500 nm HADD. Following incubation for 1 h, cells were washed, fixed, and imaged via fluorescence microscopy. Photomicrographs were taken at the exposure time determined for NA control and manipulated similarly. Bar, 10 μm. Shown are typical images from multiple fields in two different experiments. Similar results were seen in additional experiments not shown on FN^−/− cells adherent to FN or to FN lacking the N-⁹FNI region. B, dose-dependent inhibition of A488-FN incorporation into fibroblast matrices by HADD or FUD. A488-FN (20 nm) in 2% calf serum was incubated for 18 h with monolayers of human foreskin fibroblasts in the presence or absence of the indicated concentrations of FUD or HADD. Following washes in PBS, fluorescence intensity (F) was measured in a microplate reader. In the experiment shown, the fluorescence of cells not treated with A488-FN was 3900 and subtracted from each of the values. The results are typical of four experiments done with FUD and two experiments done with HADD.