Up-regulation of cell surface S100A10 expression increases plasmin generation and invasive capability of PR9 cells. S100A10-depleted PR9 cells were treated with ZnSO₄ for the indicated times and then incubated with uPA (50nM) and plasminogen (0.5μM), and the rate of plasmin generation was measured at 405 nm. Statistical analysis was performed by Student t test (***P < .001), and data are expressed as Δ405 nm/s ± SD of 3 independent experiments (A). S100A10-depleted NB4 cells, induced and noninduced, were incubated with 200nM FITC plasminogen for 1 hour, and plasminogen binding was measured by FACS. Statistical analysis was performed by one-way ANOVA with Tukey multiple comparisons, and data are expressed as the percentage of specific binding ± SD of 3 independent experiments (B). ZnSO₄-treated S100A10 shRNA-transduced PR9 cells (1 × 10⁵ cells) were added to the upper chamber of Matrigel-coated (C) or fibrin-coated (D) chambers in the absence of FBS which also contained plasminogen (0.5μM). The lower chamber contained FBS as chemoattractant. Invading cells were quantified as described in “Matrigel invasion and cell migration.” Data are expressed as mean number of cells per 40× field ± SD of 3 independent experiments. Statistical analysis was performed by one-way ANOVA (with Tukey multiple comparisons; ***P < .001). S100A10-depleted PR9 cells (5 × 10⁶), in the absence or presence of Zn²⁺, were added on top of a fibrin bed, along with 0.5μM plasminogen, and incubated overnight at 37°C. Conditioned media was collected and used for the TintElize D-dimer ELISA kit (Biopool) as per manufacturer's instructions (E).