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

Figure 4. WP1066 inhibited Stat3/VEGFR-2 signaling in brain endothelial cells.

Figure 4

(A) bEnd.3 cells were transfected with 100 nmol/L siControl or siVEGFR-2 for 48 hours in normal medium (serum free) and conditioned medium of MDA-MB-231BR cells. Protein expression of VEGFR-2, pVEGFR-2, pStat3, Stat3, and β-actin was analyzed by immunoblotting. (B) bEnd.3 cells were transfected with 100 nmol/L siControl or siVEGFR-2 for 48 hours and then subjected for tube formation assay with conditioned medium (CM) of MDA-MB-231BR cells. Lefts panels show representative experiments and right panels show quantification data (mean ± SEM) from three separate experiments. **, P<0.01; ***, P<0.001. (C) bEnd.3 cells were pretreated with a recombinant human VEGF or control vehicle in normal medium or pretreated with a VEGF neutralizing antibody (VEGF-Ab) or IgG in CM of MDA-MB-231BR cells. Then protein expression of pVEGFR-2, VEGFR-2, pStat3, Stat3, and β-actin in the cells was analyzed by immunoblotting. Right panels, quantification of pStat3 and pVEGFR-2 immunoblotting results. Each column indicates the mean ± SD from results of three different experiments. **, P<0.01; ***, P<0.001. (D) bEnd.3 cells were treated with 1 μM WP1066 in CM of MDA-MB-231BR cells for 24 hours, and whole-cell lysates were subjected to western blotting for pStat3, Stat3, VEGFR-1, VEGFR-2, and β-actin (left panel). Right panels, quantification of pStat3 and VEGFR-2 immunoblotting results. Each column indicates the mean ± SD from results of three different experiments. **, P<0.01; ***, P<0.001.