Depletion of S100A10 by shRNA in NB4 cells. NB4 cells were transduced with a retroviral shRNA system with the use of shRNA specific for 2 sequences of S100A10 (shRNA-1 and shRNA-5) and a shRNA scramble sequence (shRNA-Scr). Cell lysates were prepared, and total levels of S100A10 and annexin A2 were examined by Western blotting with the use of actin as a loading control (A). Flow cytometric analysis of cell surface S100A10 and annexin A2 (B). Quantification of flow cytometric analysis of cell surface S100A10 and annexin A2 in NB4 cells was calculated with WinMDI software (**P < .01 and ***P < .001). S100A10-depleted NB4 cells were incubated with 200nM FITC plasminogen, either with or without ϵ-ACA (100mM) or CpB (5 U/mL), for 1 hour at 4°C, 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 (C). S100A10-depleted NB4 cells were 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 (D). ATRA-treated NB4 cells (1 × 105 cells) were added to the upper chamber of Matrigel-coated (E) or fibrin-coated (F) chambers in the absence of FBS which also contained plasminogen (0.5μM). Invading cells were quantified as described in “Matrigel invasion and cell migration.” Data are expressed as the mean number of cells per 40× field ± SD of 3 independent experiments. Statistical analysis was performed by 1-way ANOVA (with Tukey multiple comparisons) in comparison to untreated NB4 cells (NT; ***P < .001).
