Figure 3.

Sp1 acted on the motif of VEGF promoter. (A) Effects of resistin on Sp1 activation in HO-8910 cells. Cells were incubated with 100 ng/mL resistin for 2 h and whole-cell lysates underwent Western blotting to detect the phosphorylation of Sp1 at either Thr-453 or Thr-739; (B) Scheme of the series of plasmid constructs containing dissected fragments of VEGF promoter; (C) Relative luciferase activity in transient transfection assays using the series of plasmid constructs (schematically shown in B) and pSp1-luciferase reporter. Relative luciferase activity was normalized with respect to the activity of PGL3-basic in untreated cells. Data are mean ± SEM from four independent triplicated experiments; (D) Chromatin immunoprecipitation of VEGF promoter complexes. HO-8910 cells underwent immunoprecipitation with anti-Sp1 antibody or control IgG. Immunoprecipitated chromatin fragments were amplified by PCR with specific primers targeting the predicted Sp1 binding motifs within the VEGF promoter; (E) Effects of various competitors in EMSA of resistin-stimulated HO-8910 cells. Competitors, including Sp1, AP-2 and Egr-1 consensus sequences, were added to the reaction mixture at a 100-fold molar excess to labeled probe, the VEGF promoter fragment containing predicted Sp1 binding motifs. Lane 2 contained no competitor. Nuclear extracts were prepared from HO-8910 cells and underwent EMSA. Cells were pre-incubated with 100 ng/mL resistin for 24 h.