Fig. 1. PAI1 increases fibronectin-matrix assembly and β1-integrin activation in MG-63 cells. (A) MG-63 cells were incubated with [¹²⁵I]fibronectin for 6 hours in the presence of the indicated concentrations of PAI1, PAI1R or PAI1 (Q123K) in DMEM supplemented with 0.02% BSA and 20 mM HEPES. Cell layers were extracted with 1% DOC, and [¹²⁵I]fibronectin that was incorporated into the detergent-insoluble matrix was recovered by centrifugation and measured by -scintillation. This experiment is representative of three separate experiments. Data are the mean ± s.e. of two experiments performed in duplicate. ^*P<0.05 (n=4), significantly different than control cells (t-test). (B) MG-63 cells were pretreated with 20 nM PAI1 for 20 minutes and incubated with 50 µM of uPAR agonist P25 or S25 for 1 hour in DMEM. Activation of β1 integrin was assessed by ELISA using the HUTS-4 antibody. Total levels of β1 integrin were measured using the P5D2 antibody against 5β1 integrin. The graph shows the levels of β1-integrin activation after normalization to total β1 levels. Neither P25 nor PAI1 had any effect on the levels of total β1 integrin. Data represent one of three different experiments performed in triplicate. ^*P<0.05 (n=3), significantly different than control or P25-treated cells (t-test).