Figure 2

Plasminogen activation system component changes on PMA-treated MCF-7 cells. MCF-7 cells were cultured for 16 hours in 5% foetal calf serum/RPMI containing 100 nM PMA. (a) Cells were washed and incubated for 30 minutes on ice with 10 to 20 μg/ml of anti-uPAR monoclonal antibody (mAb), anti-uPA mAb, anti-tPA mAb, anti-annexin II (Ann II) C-16 polyclonal antibody (pAb), anti-S100A10 (p11) pAb, or a matched isotype control antibody. Cells were then washed and incubated for 45 minutes on ice in the dark with a 1:200 dilution of appropriate secondary antibody labelled with fluorescein isothiocyanate (FITC). All cells were then washed and analysed for cell-surface-associated FITC fluorescence in the presence of 5 μg/ml propidium iodide using dual-colour flow cytometry. Specific antibody binding was calculated by subtracting control antibody fluorescence. Solid line indicates a fold increase of 1 (equivalent to no change). P values indicate a significant increase compared to the corresponding untreated control. (b) Confocal microscopy on attached cells was also used to verify enhancement of cell-surface uPAR by PMA treatment. PMA, 12-O-tetradecanoylphorbol 13-acetate; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.