Fig. 3

CGN c.3560C > T induces a mesenchymal phenotypic switch in HT-29 cells. A, Morphological change induced by 100 ng/ml EGF in CGN WT and c.3560C > T HT-29 cells. Photographs using the 40× objective. Scale bar: 20 μm. B, Western blot analyses of the expression of EMT markers in HT-29 cells with CGN WT and c.3560C > T after 100 ng/ml EGF treatment. Representative western blot from three different clones. C, (Upper) Western blot of the cytosol and nucleus β-catenin in HT-29 cells with CGN WT and c.3560C > T after 100 ng/ml EGF treatment. Lamin A and actin served as internal loading controls for cytosolic and nuclear fractions, respectively. (Lower) Densitometric quantification of the cytosol and nucleus β-catenin expression levels. Value, mean ± SEM from analysis of three different clones. D, Representative images showing E-cadherin (E-cad) (green), β-catenin (red), and DAPI nuclei (N) staining (blue) expression using immunofluorescent assay in response to 100 ng/ml EGF stimulation for 48 h. Nuclear β-catenin accumulation can be observed after EGF stimulation for 48 h in HT-29 cells with CGN c.3560C > T. Scale bar: 10 μm. E, (Left) Transwell assay and (Right) quantitative analyses of the invasion activity of CGN WT and c.3560C > T HT-29 cells. Column, mean ± SEM from analysis of three different clones. Scale bar: 200 μm. F, (Left) Migratory activities of CGN WT and c.3560C > T HT-29 cells were assessed by using gap closure assay. (Right) Quantitative analyses of the migration activity of CGN WT and c.3560C > T HT-29 cells. Gap closure area quantified by using the ImageJ software was taken as the index of cell migration activity. Column, mean ± SEM from analysis of three different clones. Scale bar: 200 μm. Data are presented as the mean ± SEM. t-test for statistical significance, *p < 0.05; **p < 0.01