Stimulation of 4T1 cells with FXa or thrombin induces uPA secretion. (A) Intracellular uPA staining. Subconfluent 4T1 cells were starved overnight then incubated with SFM, FXa, or thrombin for 30 minutes. The cells were then fixed and permeablized on glass chamber slides. Cells were incubated with both uPA-fluorescein isothiocyanate (green) and GM130-Alexa Fluor555 (red) antibodies, then counterstained with DAPI (blue). Slides were viewed on an Olympus BX51WI fluorescence microscope fitted with an Olympus DP70 cooled digital color camera. Total magnification is 400 × (10 × ocular; 40 × objective). DP Controller version 2.2.1.227 software was used for image acquisition. GraphicConverter X V5.4 was used to compile images. (B-C) Serum starved 4T1 cells were incubated with FXa (125nM) or thrombin (20nM) for 1 hour. Cellular (B) and culture supernatant (C) uPA expressed as percentage of total uPA in 4T1 cells. Results are shown as mean ± SEM of 3 independent experiments. *P ≤ .05 (control versus FXa treated) and ^§P ≤ .05 (control versus thrombin treated). (D) Cells were treated with BFA (10 μg/mL) or vehicle control then stimulated with FXa (125nM) or thrombin (20nM) for 1 hour. uPA was quantified in cell culture supernatant. **P ≤ .05 (control versus FXa treated) and ^§P ≤ .05 (control versus thrombin treated). (E) uPA levels in the culture supernatant of 4T1 cells treated with 100nM or 200nM PMA for 1 hour. *P ≤ .05 and **P ≤ .001 (control versus PMA treated). Results are shown as mean ± SEM of 3 independent experiments.