(A) Nutrient starvation causes engulfment. Graph shows the percentage of engulfment in MCF-7 cells grown in the indicated conditions for 72 hr, as determined by immunofluorescence. Glc, glucose; AA, amino acids; FBS, fetal bovine serum. Error bars depict mean ± SEM; data are from at least three independent experiments. Images show engulfed cell structures from a 72-hr glucose-starved culture. Immunostaining for β-catenin (green) and Lamp1 (red) and DAPI-stained nuclei (blue) are shown. Arrowheads show completed engulfments. Scale bar, 20 μm.

(B) Localization of cellular machinery required for entosis. Immunofluorescence image shows localization of phosphorylated myosin light chain (P-MLCS19; red, arrowhead) in MCF-7 cells grown in glucose-free conditions for 72 hr. Cell-cell junctions are shown by β-catenin staining (green, arrow), and DAPI-stained nuclei are shown in blue. The white dashed line shows the outline of partial engulfment, while the red dashed line shows the engulfed corpse. Scale bars, 15 μm.

(C) Induction of engulfment is blocked by ROCK inhibition and readdition of glucose. Graph shows the quantification of engulfment over 72 hr, as determined by time-lapse microscopy. Error bars depict mean ± SEM; data are from at least three independent experiments.

(D) Loss of E-cadherin blocks entosis. Graph shows percentage of engulfment in parental MCF-7 cells, as well as E-cad CRISPR and E-cad CRISPR + E-cad-GFP cells, grown in the indicated media conditions for 72 hr, as determined by immunofluorescence. Error bars depict mean ± SEM; data are from at least three independent experiments. Representative images show expected plasma membrane localization of exogenous E-cadherin-GFP in MCF-7 E-cad CRISPR + E-cad-GFP cells (top row) and induction of entosis in these cells (bottom row). Immunostaining for β-catenin (green) and Lamp1 (red) and DAPI-stained nuclei (blue) is shown. Scale bars, 10 μm. See also Figure S1 A.

(E) Engulfment is induced during glucose starvation in MDA-MB-231 cells expressing E-cadherin. Shown is the quantification of engulfment over 72 hr for MDA-MB-231 cells expressing either empty vector (EV) or E-cadherin-GFP, cultured in full or glucose-free media, as determined by time-lapse microscopy. Error bars depict mean ± SEM; data are from at least three independent experiments. A representative image sequence of MDA-MB-231 cells expressing E-cadherin-GFP undergoing engulfment is shown in glucose-free conditions. The red arrow shows a cell to be internalized before engulfment. White arrows show the host cell. The red arrowheads show engulfment. Time shown is in hours:minutes. Scale bars, 10 μm. Western blot shows expression of E-cadherin in parental and E-cadherin-GFP-expressing MDA-MB-231 cells.