Galectin-3-induced cell aggregation enhances cell survival by avoiding initiation of anoikis. A: Galectin-3-induced cell aggregation is associated with increased survival of the cells under anchorage-independent conditions. HT29-5F7 cells treated with or without 1 μg/ml galectin-3 or BSA for 1 hr followed by culture of the cells in suspension for 3 days at 37°C. After separation of the cells by cell strainers, the viability of the strained (single) cells and cell aggregates was assessed. The data are presented as mean ± SEM of triplicate determinations from two independent experiments. B: The presence of 20 μg/ml anti-E-cadherin antibody inhibited galectin-3-mediated increase of survival of HT29-5F7 cell aggregates. The data are presented as mean ± SEM of triplicate determinations from two independent experiments. C and D: Galectin-3 treatment has no effect on anoikis of the single cells (C) but significant reduction of anoikis of the cell aggregates (D). Anoikis was assessed by Annexin-V cell surface binding after treatment of the cells with galectin-3 and subsequent separation of the single cells and cell aggregates. The data are presented as mean ± SEM of triplicate determinations from two independent experiments. E. Representative flow cytometry plots from the anoikis assessments of HT29-5F7 cells in the presence of 1 μg/ml galectin-3 or BSA for 48 hr at 37°C. Annexin-V positive and PI negative (early apoptotic, at the bottom right in the bivariate correlation plot) and Annexin-V positive and PI positive (late apoptotic, at the top right in the correlation plot) cells are considered as apoptotic cells.