Overexpression of ENO-1 by U937 cells increased cell-surface plasmin generation and enhanced directed migratory, transmigratory, and invasive properties. (A) ENO-1 expression was examined in stably transfected U937 cells overexpressing ENO-1 wild-type (ENO-1WT) or ENO-1 lacking the plasminogen binding site (ENO-1ΔPLG), as well as in mock-transfected (pcDNA3.1) or native (−) cells. The ENO-1 located in the cytosol (left panels) or associated with the cell membrane (right panels) was determined by Western blot. The purity of cytosolic and membrane fractions was assessed by probing the samples for CD18 and P20S, respectively. The Western blot illustrated is from 1 representative experiment of 4. (B) Comparison of ENO-1 cell-surface expression by U937 stable transfectants using flow cytometry. (Left panel) Unstimulated cells transfected with pcDNA3.1 (thin line) versus ENO-1WT (bold line); (middle panel) ENO-1WT–transfected cells stimulated with LPS (5 μg/mL, 6 hours; bold line) versus unstimulated (thin line); (right panel) pcDNA3.1-transfected cells stimulated with LPS (5 μg/mL, 6 hours; bold line) versus unstimulated (thin line). Isotype controls are represented by the shaded histograms. One representative experiment of 3 is illustrated. (C) Plasmin proteolytic activity as measured by S-2251 degradation by stably transfected U937 cells as indicated, either in the absence or presence of LPS (5 μg/mL LPS, 6 hours). Data represent mean values ± SD from 4 independent experiments, each performed in quintuplicate. Directed migration (D), transmigration (E), and invasion (F) of unstimulated stably transfected U937 cells toward the chemokine MCP-1 (■), compared with basal activity in absence of MCP-1 (□). The experiments were conducted in analogy with those described in Figure 4. Data represent mean values ± SD from 4 independent experiments, each performed in quintuplicate. NS indicates not significant; *P < .05; **P < .01.