Figure 5

Plasminogen binding and activation on MCF-7 cells. After culture for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA (PMA-stimulated) or vehicle alone (control), cells were detached and pre-incubated for 10 minutes at room temperature in the absence or presence of 50 nM active urokinase plasminogen activator (uPA) or PMSF-inactivated uPA (PMSF-uPA), washed, and then analysed for cell-surface plg binding or activation. (a) Cell-surface, lysine-dependent plg binding to PMA-stimulated MCF-7 cells is shown as a percentage increase compared to unstimulated MCF-7 cells in the absence of uPA. (b) Cell-surface plasmin (pln) generation. Pln activity assays were performed using Spectrozyme PL in the presence of α₂-antiplasmin to inhibit any solution-phase pln generation. Activity in the presence of aprotinin (pln inhibitor) was also measured and subtracted from all values to determine pln-dependent activity. (c) Cell-surface lysine-dependent fluorescein isothiocyanate-plg binding was measured in the presence or absence of aprotinin. Percentages show the proportion of binding due to pln activity at the cell surface (that is, pln-dependent binding calculated as total binding minus binding in the presence of aprotinin; open bars) and the proportion that is independent of pln activity (that is, pln-independent binding calculated as residual binding in the presence of aprotinin; hatched bars), which together constitute total lysine-dependent plg binding. *Significant increase compared to unstimulated control cells not pre-incubated with uPA or PMSF-uPA (p < 0.05). **Significant increase compared to PMA-stimulated cells not pre-incubated with uPA or PMSF-uPA. MFI, mean fluorescence intensity; PMA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, alpha-toluenesulfonyl fluoride.