FIGURE 4.

FoxC2 binding to the FOX:ETS motif is decreased. A, shown is a representation of VEGFR-2 promoter deletion mutants used in our studies. B, VEGFR-2 promoter activation was unaffected by deletions. Different VEGFR-2 promoter deletion-mutant luciferase constructs with Renilla luciferase vector and control or VEGF-A siRNA were co-transfected into HUVECs. Forty-eight hours later, firefly and Renilla luciferase activities were measured. C, binding of FoxC2 to the FOX:ETS motif was decreased by endogenous VEGF-A knockdown. Forty-eight hours after siRNA transfection, HUVECs were subjected to chromatin immunoprecipitation by anti-FoxC2 antibody. Immunoprecipitated DNA fragments were used to perform PCR. D, pGL3-promoter empty vector or containing VEGFR-2 enhancer constructs with Renilla luciferase vector and control or VEGF-A siRNA were co-transfected into HUVECs. Forty-eight hours later, firefly and Renilla luciferase activities were measured. E–G, VE-cadherin and Tie-2 levels were also down-regulated. The same procedure as for VEGFR-2 in Fig. 3 was followed. All bars represent means ± S.D. of three experiments. *, p < 0.05.