Fig. 1. Entosis in MCF7 breast epithelial cells after glucose deprivation.

A Confocal images of entosis events in wild-type MCF7 cells grown for 72 h in 0 mM glucose media. Cells were stained with antibodies against E-cadherin (violet), LAMP1 (red) and β-catenin (green). Nuclei were stained with Hoechst (blue). B Percentage of entosis in wild-type MCF7 cells after manual counting of cell-in-cell bodies representing entotic/engulfed cells at the 72 h time point at 0 mM glucose. Entotic cells were determined using confocal imaging (Two-tailed t-test; n = 5). C Representative confocal photographs of engulfed cell-in-cell bodies identified as entotic bodies for manual counting. Nuclei were stained with Hoechst (blue). D Percentage of apoptotic+necrotic cells in wild-type MCF7 cells after 72 h of glucose deprivation in the presence of ROCKi (Y27632, 10 µM) or vehicle (DMSO). Cells were analyzed by flow cytometry using Annexin V-APC and SYTOX™ Green Nucleic Acid Stain (Two-tailed t-test; n = 3). E Percentage of entotitic cells observed in wild-type MCF7 cells at the 72 h time point of growth in 0 mM glucose media in the presence or absence of NAG (15 mM). Entotic cells were determined using confocal imaging and manually counted (Two-tailed t-test; n = 3). F Western blot for human LAMP1, E-cadherin and Concanavalin A after culturing the cells at 0 mM glucose, or 0 mM glucose in the presence of NAG (15 mM).