Galectin-3 and B27.29 anti-MUC1 mAb both induce increase of cell aggregation. A: B27.29 mAb induces dose-dependent increase of HCA1.7+ cell aggregation. HCA1.7+ cell aggregation was determined after pre-incubation of the cells with or without various concentrations of B27.29 Mab, 1 μg/ml control mouse immunoglublin or CT-2 anti-MUC1 antibody. Data are expressed as mean ± SEM of triplicate determinations from three independent experiments. B: B27.29 mAb increases HCA1.7+ cell aggregation regardless of the presence or absence of recombinant galectin-3. HCA1.7+ cell aggregation was determined after pre-incubation of the cells with or without 1 μg/ml recombinant galectin-3 in the presence or absence of 1 μg/ml B27.29, BSA or control immunoglublin. Data are expressed as mean ± SEM of triplicate determinations from three independent experiments. C: Galectin-3 or B27.29 mAb at 1 μg/ml fails to induce HCA1.7+ cell aggregation at 4°C or to paraformaldehyde-prefixed cells. Data are expressed as mean ± SEM of triplicate determinations from four independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001. D: MUC1 localization in cell aggregates. Separate aliquots of HCA1.7+ cells pre-labelled with DiO (green) or DiI (red) were mixed in the presence of 1 μg/ml galectin-3 or B27.29 mAb and incubated for 1 hr at 37°C before fixation and subsequent analysis of MUC1 localization by immunohistochemistry. Representative images of the MUC1 localization in cell aggregates are shown. F: localization of the cell aggregates-associated MUC1 (green) after treatment of the cells with 1 μg/ml galectin-3 for 48 hr under suspension (red: cell nucleuses). Bar = 10 μm.