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. 2015 Mar 12;6(12):10016–10029. doi: 10.18632/oncotarget.3540

Figure 5. Stat3 signaling regulates VEGFR-2 expression in bEnd.3 cells.

Figure 5

(A) bEnd.3 cells were transfected with 100 nmol/L of siRNA (siControl or siStat3) or 6 μg of cDNA plasmid (vector or Stat3C) for 48 hours with serum-free normal medium (NM) or conditioned medium of MDA-MB-231BR cells (CM). Protein expression of Stat3, VEGFR-2, and β-actin was analyzed by immunoblotting. (B) Schematic structure of the murine VEGFR2 promoter with the sequences of Stat3-binding sites. (C) ChIP assays in bEnd.3 cells cultured with CM of MDA-MB-231BR cells. Chromatin fragments of the cells were immunoprecipitated with Stat3 antibody or IgG and subjected to PCR using primers for both Stat3-binding sites. (D) Stat3 transactivates VEGFR-2 promoter. Left, bEnd.3 cells were transfected with the murine VEGFR2 promoter and Stat3C or control vector (6 μg) for 48 hours in NM. Luciferase activities of the cells were then determined. Right, bEnd.3 cells were transfected with the murine VEGFR2 promoter and siStat3 or siControl (100 nmol/L) for 48 hours in CM of MDA-MB-231BR cells or treated with WP1066. bEnd.3 cells transfected with the murine VEGFR2 promoter in serum-free normal medium (NM) served as a control. Luciferase activities of the cells were then determined.