Ezrin KD blocks Stat3 phosphorylation, VEGF-A/-C, and IL-6 expression. Representative western blots display pY705-Stat3, Stat3, and survivin (panel A) and VEGF-A/-C (panel B) levels in MDA231, MDASrc, and MDASrc shEZR lysates. γ-tubulin expression was used as loading control. Densitometry results are presented as an average (n = 3) ratio of target protein to γ-tubulin and normalized against MDA231 controls (bar graphs). P values were calculated using unpaired t test. (C) Cell lysates from MDA231 cells expressing the empty vector pCB6 or wild-type ezrin (EZR/WT) were analyzed for VEGF-A, pY705 Stat3, and Stat3 levels. (D) Immunoblot analysis of IL-6 in cell lysates from MDA231 and MDASrc cells expressing the empty vector pLKO.1 or shEZR vector. (E) Immunoblot analysis of IL-6 levels in cell lysate from MDA231 cells expressing the empty vector pCB6 or wild-type ezrin (EZR/WT). (F) Immunoblot analysis of VEGF-A, pY705 Stat3, and Stat3 in MDA231 shEZR cells treated with vehicle alone or 20 ng/ml recombinant human IL-6 for 20 hr. (G) The angiogenic activity of CM collected from confluent MDASrc in the presence of α-IL-6 neutralizing antibody or non-immune IgG was assessed using the aortic ring assay. Endothelial cell sprouting was quantified using ImagePro software and presented as percentage of control (bar graph). Identical amount of α-IL-6 antibody was added to rings in the MDASrc group to control for the direct effect of antibody on endothelial cell sprouting. Panels C-F: relative densitometry values are displayed under corresponding bands (normalized against γ-tubulin). CM: conditioned media; IgG: immunoglobulin G; IL-6: interleukin-6; KD: knockdown; Stat3: signal transducer and activator of transcription 3; VEGF: vascular endothelial growth factor.