SHP-1 is a crucial mediator of endorepellin antiangiogenic effect. (A) Immunoblot of HUVECs from either mock transfected or transfected with a pool of 3 different SHP-1 siRNAs. Notice the marked knockdown of SHP-1 expression, whereas SHP-2 levels are unaffected. (B) Immunoblot of HUVEC lysates after SHP-2 knockdown by siRNA. (C) Quantification of VEGF-directed migration of HUVECs through collagen type I. Control untreated cells or cells with SHP-1 knockdown, and cells treated with Na₃VO₄ (1μM) or NSC-87877 (1 μM) were incubated with or without 25nM endorepellin for 20 minutes before commencement of the migration assay. Values represent means ± SEMs (n = 3); ***P < .001. (D) Quantification of migration assays as in panel C. Note that the cells show a slower migration after SHP-2 knockdown, but they still respond to endorepellin; means ± SEMs (n = 3); ***P < .001. (E) Fluorescent images of endothelial cells incubated with rhodamine phalloidin to visualize the actin cytoskeleton (red stress fibers) and DAPI to visualize the nuclei (blue). Notice that the disassembly of actin cytoskeleton after a 20-minute treatment with 25nM endorepellin (ER). This process is blocked by Na₃VO₄ and NSC-87877. Scale bar, 10 μm. (F) Quantification of the number of actin filaments per cell (n = 60 for each group); ***P < .001.