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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Nat Cell Biol. 2011 Oct 16;13(11):1335–1343. doi: 10.1038/ncb2363

Figure 1.

Figure 1

Entotic cell death involves recruitment of LC3 to a single-membrane vacuole. (a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP-LC3 and H2B-mCherry. Arrow marks recruitment of LC3 to the entotic vacuole, see Supplementary Information Movie S1. Bar = 15µm. (b) Images of MCF10A expressing GFP-LC3G120A and mCherry-LC3; note no recruitment of GFP-LC3G120A to entotic vacuole. Bar = 15µm (c) LC3 recruits from host cell cytosol onto the entotic vacuole membrane. Data points are mean fluorescence intensity of GFP-LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures, see Supplementary Information Movie S2. (d) Correlative video-light electron microscopy of MCF10A GFP-LC3 cell-in-cell structure. Cells were imaged by time-lapse, followed by fixation after LC3 recruitment, see Supplementary Information Movie S3. Post-fixation images (top panels) of phase and GFP were taken demonstrating LC3 recruitment. Electron microscopy of the same cell shows the entotic vacuole is a single membrane (arrow). (e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of internalized cell. Data represent mean +/− SEM from at least 3 independent experiments, n=total number of death events. *P<0.03, ***P<0.0001. Time-lapse images of MCF10A cell-in-cell structures expressing either: (f) 2xFYVE-mCherry (red) and GFP-LC3 (green), or (g) Lamp1-GFP (green) and mCherry-LC3 (red). Arrows indicate recruitments to the entotic vacuole, see Supplementary Information Movies S4 and S5. Times are indicated as hr:min:sec (f) or min (g). Bar = 5µm. (h) Representative images of cell-in-cell structures where only host cells express CathepsinB-mCherry. Live inner cell with no CathepsinB in the vacuole, arrow (left panel), live internalized cell with CathepsinB from the host inside of the vacuole, arrow head (middle panel), dead internalized cell with CathepsinB from the host throughout the corpse, asterisk (right panel). Bar = 15µm. (i) Five representative timings of GFP-LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (X) from time-lapse microscopy. (j) Initiation and duration of 2xFYVE domain (red bar, n=11), LC3 (green bar, n=20) and Lamp1 (orange bar, n=7) recruitment to entotic vacuoles, determined using double expressing cells shown in (f, g). Error bars represent SD.