muPAR expression in 293 cells induces VN-dependent ERK1/2 activation and cell growth. (A) muPAR expression induces VN-dependent ERK1/2 activation. Semiconfluent cells were serum starved for 4 hours prior to cell lysis and immunoblot analysis of ERK1/2 activation. Blots were probed for phosphorylated (Thr²⁰²/Tyr²⁰⁴) ERK1/2, stripped, and reprobed for total ERK1/2. Representative blots are shown, and the quantification of independent (n = 5) experiments is shown (bar graph). (B) uPAR promotes VN-dependent cell growth of HEK293 cells. Cells were seeded in 96-well tissue culture plates. Cell growth was quantified by measuring the relative increase in GFP-fluorescence. (C) Effect of uPAR–VN interaction on cell proliferation. Semiconfluent cells were pulsed with BrdU for 1 hour and stained with an anti-BrdU antibody and PI. BrdU incorporation and DNA content were analyzed by flow cytometry. The graph represents the mean of percentages of cells in S phase according to BrdU signal and DNA content (n = 4). (D) Effect of uPAR–VN interaction on apoptosis. Cells were stained with an annexin V antibody and PI prior to analysis by flow cytometry. Representative dot plots of 3 independent experiments are shown. The global cell population was divided into 4 subpopulations according to the PI and annexin V signal intensity. The percentage of double-positive apoptotic cells is shown.