Involvement of cell surface E-cadherin in galectin-3-induced cell aggregation. A: The presence of anti-E-cadherin antibody reduces spontaneous aggregation of HT29 but has little effect on aggregation of HT29-5F7 cells. Data are expressed as mean ± SEM of triplicate determinations from three independent experiments. B: Anti-E-cadherin antibody at 20 μg/ml prevents HT29-5F7 cell aggregation-induced by (1 μg/ml) galectin-3. Data are expressed as mean ± SEM of triplicate determinations from three independent experiments. C: E-cadherin localization in single cells and cell aggregates. HT29-5F7 cells released by NECDS and labelled separately with DiO and DiI were mixed in the presence or absence of galectin-3 for 1 hr at 37°C. The cells were fixed, probed with mAb B27.29 anti-MUC1 or anti-E-cadherin and fluorescent-labelled secondary antibody and analysed by fluorescent microscopy. E-cadherin shows localization/accumulation at the cell-cell contact points (arrowed) in the cell aggregates. D: Western blot showing that transfection of HT29-5F7 cells with siRNA for E-cadherin suppresses E-cadherin expression. E: E-cadherin immunohistochemistry shows marked reduction of the cell surface E-cadherin (green) in HT29-5F7 cells after treatment with E-cadherin siRNA for 72 hr (red: cell nucleus). F: SiRNA-mediated knock-down of E-cadherin expression prevents galectin-3-mediated HT29-5F7 cell aggregation. Data are expressed as mean ± SEM of triplicate determinations from three independent experiments. G: flow cytometry analysis of HT29 and HT29-5F7 cells stained with the MAB1838 anti-E-cadherin antibody show similar E-cadherin cell surface expressions. Open histogram: E-cadherin; shaded histogram: immunoglobulin isotype control.