Figure 2

AGS8 knockdown inhibited VEGF-induced cell proliferation and cell migration in choroid endothelial cells. (A) MTT assay of AGS8 knockdown in VEGF-stimulated cells. After transfection of control siRNA or AGS8 siRNA#1, cells were stimulated by VEGF at 10 ng/mL for 48 h and cell proliferation was analyzed with MTT assay. Data are means ± s.e.m. from 6 independent experiments repeated 4 times. ^**P < 0.01 (two-way ANOVA with Tukey’s correction). N.S., not significant (unpaired t-test). (B) Transwell migration analysis of AGS8 knockdown in VEGF-stimulated cells. Cells transfected with AGS8 siRNA#1 were seeded in transwells, stimulated by VEGF at 10 ng/mL for 4 h, and stained. Images of migrated cells were taken in three random microscopic fields. Data are means ± s.e.m from four independent triplicate experiments. ^**P < 0.01 (two-way ANOVA with Tukey’s correction). N.S., not significant (unpaired t-test). (C) VEGFR-2 expression on the cell surface is reduced by AGS8 knockdown. RF/6A cells were transfected with control siRNA or AGS8 siRNA#1, and labeled with an fluorescein-conjugated anti-VEGFR-2 antibody, and flow cytometric analyses were performed as described in the Methods. The histograms represent cell counts (Y-axis, linear scale) versus fluorescein intensity (X-axis, log scale). Negative control indicates negative control cells treated with respective isotype human IgG labeled with fluorescein. Fluorescence mean intensity obtained from the histograms was quantified and shown in bar graphs (right panel). Data are the mean ± s.e.m from 4 experiments. ^**P < 0.01 versus control siRNA-treated cells (unpaired t-test).