Figure 5.

Effect of stable PAI-1 on ECM degradation by transfected NS0 cells expressing ch-uPA_RRHR and ch-uPA_wt. Transfected or untransfected NS0 cells were seeded (2 × 10⁶ cells/2 ml) onto ³⁵S-labeled ECM (9 × 10⁵ cpm/35-mm dish) in the absence or presence of recombinant, stable PAI-1 (31), or benzamidine (benz.). The cultures were incubated at 37°C in serum-free growth medium and at the indicated times, 200 μl of the medium was removed and centrifuged to remove cell debris, and the supernatant was counted. The total radioactivity released into the culture supernatant was calculated for each time point after subtracting the cpm released from the control dishes incubated in the absence of cells. The radioactivity released from the ECM in the absence of cells totaled 5–10% that in the presence of untransfected NS0 cells (0.04–0.20 × 10⁵ cpm/35-mm dish). (A) ECM degradation mediated by untransfected NS0 cells or NS0 cells transfected with and expressing the ch-uPA mutant (NS0-ch-uPA_RRHR). Recombinant, stable PAI-1 was added to the cultures at the indicated concentrations. (B) Degradation of radiolabeled ECM by NS0 cells transfected with and expressing wt ch-uPA (NS0–ch-uPA_wt). Recombinant stable PAI-1 or benzamidine was added to the cultures as indicated.