Role of IL-6 in aggressive tumor behavior and EMT changes. a. Effect of IL-6 inhibition on the migration ability of esophageal cancer cells. Plates were photographed at the times indicated. Representative slides and quantitative data (y-axis shows the relative ratio, normalized to the distance under control conditions) are shown. b. Effect of IL-6 inhibition on EMT-associated proteins. The changes of E-cadherin and vimentin expression were evaluated by immunofluorescence, and the results of representative slides are shown. In addition, changes in the levels of EMT-associated proteins were evaluated by immunoblotting (W, wild-type cells; V, cells transfected with the control vector; IL-6-, cells transfected with the IL-6 silencing vector). c. Representative IHC staining with an anti-p-STAT3 antibody in esophageal cancer and adjacent non-malignant epithelium on slides from TMA blocks. d. P-STAT3 levels correlate positively with IL-6 levels in human esophageal cancer specimens (p < 0.001). Representative images of positive IL-6 and p-STAT3 staining on slides from a selected tumor specimen, and representative negative staining for IL-6 and p-STAT3 on slides from another tumor specimen, are shown. e. Effect of the IL-6 silencing vector on STAT3 activation, as examined by immunofluorescence analysis. f. Effect of inhibited IL-6 signaling on STAT3 activation and EMT-related protein levels, as determined by immunoblotting (W, proteins were extracted from cells under control condition; IL-6Ab, proteins were extracted from cells incubated in the presence of 5 μg/ml IL-6 neutralizing antibodies for 24 h; AG, proteins were extracted from cells incubated in the presence of 50 μM AG490 for 24 h; SI, proteins were extracted from cells 72 h after transfection with STAT3 siRNA).