Effect of PAI-1 on P25 induced integrin activation and matrix assembly. A – MG-63 cells monolayers, grown to confluency in 12-well plates, were washed and pretreated with different concentrations of PAI-1 for 20 minutes in the presence of 125I-fibronectin and then stimulated with 50 μM S25 or P25 for 6 hours. Cells were rinsed, scraped directly into 1 ml of 1% deoxycholate and the total cell bound 125I-fibronectin was counted. B, C – MG-63 cells, grown to confluency in 24-well plates, were washed, pretreated with different concentrations of PAI-1 for 20 minutes and incubated for 1 hour in DMEM containing 50 μM P25 or S25. Cells were then incubated for 1 hour with 100 ng/ml of either monoclonal antibody, HUTS-4 (B) or P5D2 (C). Cell layers were washed, fixed in paraformaldehyde, and incubated with a peroxidase conjugated secondary antibody. Bound antibody was detected by incubating cells with substrate, and color development was measured at A490 in a plate reader.