Effect of KIAA1199 expression on angiogenesis. (
) Cell migration assay. Human umbilical vein endothelial cells (HUVEC) transfected with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 were plated onto the top of the transwell, allowed to migrate for 4 h, then rinsed, fixed, stained, and counted. Left panel: HUVEC on the undersurface of a filter, magnification × 40; right panel: number of HUVEC per field. (B) Tube formation assay: 24-well plates were pre-coated with Matrigel (diluted 1:2 in H2O) and incubated at 37°C to promote gelling. HUVEC were seeded on Matrigel-coated plates, treated with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199 for 6-8 h, tube formation observed using a phase-contrast inverted microscope, and photographs were taken from each well. The number of intersections between branches of assembled endothelial cell networks was counted in the whole field. (
C). Chorioallantoic membrane (CAM) assay, embryonic CAM was treated on day 7 with scrambled siRNA, KIAA1199 siRNA duplex, pcDNA3.2DEST and pcDNA3.2DEST-KIAA1199. After incubation for 3 days CAM was examined under a stereomicroscope, and photographs taken (left panel). ImageJ 2.43 s was used to calculate the vascular and CAM areas. Bar graph: ratio of vascular area to CAM area (right panel). All experiments were performed at least in triplicates, values are presented as mean ± SD; *P <0.05 compared with control (*significant differences).