Effect of ECM molecules on HGF induced Endothelial cell migration. Panel A-Dye-loaded HMVEC suspensions (9 × 104/ml) were treated with HGF (10 ng/ml) in the presence or absence of ECM molecules, FN or VN or collagen-1 in a modified Boyden chamber assay. Migration of cells was measured at 3 hours. The data is presented as relative migration with the migratory response presented as ratio to the basal migration in the absence of stimulus. Panel B-The levels of cell adhesion to Fluorblok transwell filters coated with either poly-L-lysine (PLL) or the various ECM glycoproteins was determined. Dye loaded HMVEC (105 cells) were treated with HGF (10 ng/ml) for 30 min prior to application to upper chamber of the transwell. Following extensive washes the number of adherent cells was determined using a fluorescence plate reader. The role and identity of integrins mediating the migration response in cells stimulated with HGF and FN (panel C) and HGF and VN (panel D) was demonstrated by the pre-treatment of HMVEC suspensions with anti-integrin monoclonal reagents (10 μg/ml) with specificity for integrins α5β1 (JBS5), αvβ3 (LM609) and αvβ5 (LM142) for 30 min at room temperature prior to application into the upper transwell chamber. The data is presented as specific migratory response in fluorescence units with the basal migration subtracted from the total migratory response. (n = 2).