Effect of Zf9 on endogenous PA activity in BAEC cultures.

(A) Effect on mRNA expression of uPA, uPAR, and PAI-1. BAEC cultures grown on 10-cm dishes were transfected with 8 μg ofZf9-pCIneo. As a control, cells were transfected with the same amount of pCIneo. Forty-eight hours after transfection, cell lysates were prepared, total RNA was extracted, and mRNA expression of uPA, uPAR, PAI-1, and GAPDH in each sample was assessed by Northern blotting. Radioactivity of each band was detected on an image analyzer, and relative intensity of each band was quantitated, normalized against the intensity of GAPDH, and expressed as arbitrary units in parentheses under each band. Each experiment was repeated 3 times with similar results in all, and representative results are shown. (B) Effect on BAEC PA activity levels. BAEC cultures grown on 35-mm dishes were transfected with the indicated amounts of Zf9-pCIneo. Forty-eight hours after transfection, cells were harvested in lysis buffer, and PA levels in each lysate were determined using chromogenic substrate, S-2403, and expressed as urokinase (UK) unit (U)/mg of protein in the sample. Each value represents the average ± SD from triplicate determinations. Points marked by an asterisk differ significantly (P < 0.05) from control cells transfected with 2 μg of pCIneo alone. Experiment was repeated 4 times with similar results in all, and representative results are shown.