Characterization of elevated expression or activity of the uPA signaling pathway in highly invasive breast cancer cells. (A) Bar graph showing the comparable levels of mRNA transcript expression for uPA, uPAR, and its endogenous inhibitors PAI-1 and PAI-2 between MDA-MB-231Hi and parental MDA-MB-231 cells. (B) Immunoblots showing the elevated uPA, uPAR, and PAI-1 expression and decreased PAI-2 expression in MDA-MB-231Hi cells relative to MDA-MB-231 cells. The blots were reprobed with β-tubulin as a loading control. (C) Flow cytometry confirming increased cell-surface uPAR expression in MDA-MB-231Hi cells (68.08% ± 3.770% shift relative to 40% ± 2.492% in MDA-MB-231 cells; P < 0.01; n = 3). Next, activity assays were used to detect changes in uPA activity, measured within a "cell-associated" fraction (D) and within the supernatant (E) of the cell culture. Activity of this serine protease was significantly higher in both the cell-associated fraction (2.64-fold increase; P < 0.01; n = 4) and the supernatant (2.56-fold increase; P < 0.01; n = 4) of the MDA-MB-231Hi cells compared with the parental MDA-MB-231 cells. (F) An increased level of plasmin activity was observed in the MDA-MB-231Hi cells relative to parental cells, conducted over a 3-hour period (P < 0.05; n = 4). Statistically significant differences in all quantitative assays were determined by using a Student two-tailed t test (*P < 0.05; **P < 0.01; ***P < 0.001).