Treatment of endothelial cells with rhaPC prevents B16-F10 adhesion and transmigration. (A) Flow cytometric analysis of EPCR expression in bEnd.3 and B16-F10 cells. EPCR staining (solid line) performed with FITC-labeled anti–mouse mAb (clone RMEPCR1560; StemCell Technologies, Vancouver, BC). Isotype control (IgG2b) is represented by the shaded area. (B) Confluent bEnd.3 monolayers were pretreated with rhaPC (0, 5, 10, 25, 100 nM) for 3 hours and washed. CFDA-SE–labeled B16-F10 melanoma cells were then added to each well. After 1 hour, wells were washed and adherent fluorescent cells were counted. ***P < .001, **P < .01 compared with untreated group. Each dose was tested in quadruplicate. Data are representative of 3 independent experiments. (C) Confluent bEnd.3 monolayers on 8-μm pore Transwell inserts were pretreated with rhaPC for 3 hours. Chambers were washed, and DMEM was supplemented with 15% FCS added to each lower chamber. CFDA-SE–labeled B16-F10 melanoma cells were added to the upper chamber. Cells in the lower chamber were counted 16 hours later. ***P < .001, compared with the untreated group. Each dose was tested in quadruplicate. Data are representative of 3 independent experiments. (D) CFDA-SE–labeled B16-F10 melanoma cells were pretreated with rhaPC for 3 hours, washed, and then applied to confluent bEnd.3 monolayers. After 1 hour, wells were washed thoroughly and adherent fluorescent cells were counted. Each dose was tested in quadruplicate. Data represent 3 independent experiments. (E) B16-F10 melanoma cells were treated with rhaPC (0, 5, 10, 25, 100 nM) 5 times at 1-hour intervals and assessed for viability by MTT assay. Each dose was tested 16 times per experiment. Data are representative of 3 independent experiments. All data reported as mean with standard error.
