Figure 4. Kp-10 decreases Sp1-dependent VEGF expression.

(A) Kp-10 inhibits VEGF expression in HUVECs. HUVECs were treated with different concentrations of Kp-10 for 24hrs and then subjected to RT-PCR and Western blot analysis with anti-VEGF antibody. GAPDH and actin were used for PCR and Western blot loading controls, respectively. (B) Kp-10 mediates the inhibitory effect through the GC-rich boxes in VEGF-promoter regions. (Left) HUVEC transfected with pVEGF₂₀₁₈-luc was treated with CoCl₂ or HIF1 α in the presence or absence of 1 μM of Kp-10. At 24hr, luciferase assays were performed. *, p<0.05. (Middle and right) HUVEC was transfected with pVEGF₁₃₃-luc or pVEGF₈₅-luc plasmid, and then treated with different concentrations of Kp-10 (10⁻², 10⁻¹, 1, and 10 μM) for 24hrs before luciferase activity was examined. *, p<0.05; **, p<0.01. (C) Kp-10 inhibits the DNA binding activity of Sp1 protein to VEGF promoter region in EMSA assays. Kp-10 inhibits both hypoxia-independent and dependent Sp1 DNA-binding activity to VEGF promoter. When HUVEC was exposed to 1 μM of Kp-10 for 24hr, the binding of Sp1 to VEGF promoter region was diminished. The effects of Kp-10 on Sp1 DNA binding of VEGF promoter in hypoxic condition were measured with the addition of 100nM of CoCl₂. (D) Kp-10 inhibits DNA binding activity of Sp1 to VEGF promoter in vivo. ChIP assays were performed with different concentrations of Kp-10 after the cells were treated for 24 hr.