The effect of HWF on EMT markers and STAT3 as a mediator of the HWF effect on HN-7 cells. (a) Cell lysates prepared from HN-7 cells incubated with 10% FBS or HWF for 24 or 72 h were subjected to western blotting (6.4 μg protein per lane) with antibodies against E-cadherin, β-catenin, and S100A4. The band intensities were quantified with the AlphaView software package and normalized by Coomassie staining. The expression is displayed in relation to the expression in the FBS-treated controls. The experiment was repeated three times with similar results. (b) Western blot of lysates (5.6 μg protein per lane) from cells grown with 10% FBS or HWF for 24 h (upper panels). The lower panel shows the loading control adjusted expression in the cells grown with HWF compared with the expression in FBS-treated cells. (c) Inhibition of STAT3 phosphorylation by S3I-201. HN-7 cells were grown with 10% FBS, HS, or HWF and the indicated concentrations of S3I-201. Phospho-STAT3 was analyzed by western blotting (upper panel) and quantified in relation to the loading control (lower panel). (d) Growth inhibition of HN-7 cells grown with 10% HS or HWF. EC50 was 98 μmol/L and 61 μmol/L in the presence of HS and HWF respectively (N=6). The maximum difference in inhibition was at 100 μmol/L S3I-201. (e) The effect of S3I-201 on migration analyzed by the scratch assay (N=16). The HWF and HS/FBS results were from different experiments but normalized by inclusion of samples measured without S3I-201 addition in both experiments. Several experiments including all medium supplements but with fewer S3I-201 concentrations yielded similar results. Error bars represent SEM.