Figure S5.
Elimination of epithelial cells dying by apoptosis, pyroptosis, or necroptosis. (A) Representative time-lapse images of opto-hCaspase-8-transgenic Caco-2 cells undergoing apoptosis in absence of neighboring cells. Annexin V staining (blue) marks PS exposure during late timepoint. Arrows indicate characteristic apoptotic features: initial cell contraction (15 min), cell shrinking and detachment (30 min), rounding up and PS exposure (60 min). (B) Absence of apoptotic body formation in isolated Caco-2 cells versus formation of apoptotic bodies in MDCK cells. (C) Percentage of fragmented apoptotic cells in opto-hCaspase-8-transgenic Caco-2 cells cultured either alone (monoculture) or together with WT Caco-2 and stimulated with blue light for 1 h. (D) Percentage of extruded, partially extruded apoptotic cells in co-cultures treated with Annexin-V (10 µg/ml). (E) Representative time-lapse images of spontaneous apoptosis and apoptotic corpse efferocytosis in wild-type Caco-2. Scale bars, 20 µm. (F) Representative images of lifeact-GFP distribution in non-stimulated Caco-2 cells. Basal plane (0 µm) shows randomly oriented lamellipodia, and medial plane (+5 µm) shows cortical actin enrichment at the cell junctions). (G) Representative time-lapse images of actin rearrangement in neighboring cells during apoptotic cell extrusion. Scale bar, 20 µm. Cells were stimulated and images were acquired as in Fig. 8, A–C. (H) Reconstructed side view (xz) of time-lapse series from B. Arrows indicate incomplete phagocytic cup formation during failed corpse engulfment. (I) Percentage of extruded cells over time. Cell death was induced by illumination as described previously in the co-cultures treated with the indicated inhibitors. The time of extrusion was determined by complete gap closure in the imaging plane, determined by the CellMask signal. Data are pooled from three (C) or two (D) independent experiments (total number of cell death events as indicated). Data in I are pooled from two independent experiments (total number of cell death events as indicated in Fig. 9 H. Mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Scale bars, 20 µm.

Elimination of epithelial cells dying by apoptosis, pyroptosis, or necroptosis. (A) Representative time-lapse images of opto-hCaspase-8-transgenic Caco-2 cells undergoing apoptosis in absence of neighboring cells. Annexin V staining (blue) marks PS exposure during late timepoint. Arrows indicate characteristic apoptotic features: initial cell contraction (15 min), cell shrinking and detachment (30 min), rounding up and PS exposure (60 min). (B) Absence of apoptotic body formation in isolated Caco-2 cells versus formation of apoptotic bodies in MDCK cells. (C) Percentage of fragmented apoptotic cells in opto-hCaspase-8-transgenic Caco-2 cells cultured either alone (monoculture) or together with WT Caco-2 and stimulated with blue light for 1 h. (D) Percentage of extruded, partially extruded apoptotic cells in co-cultures treated with Annexin-V (10 µg/ml). (E) Representative time-lapse images of spontaneous apoptosis and apoptotic corpse efferocytosis in wild-type Caco-2. Scale bars, 20 µm. (F) Representative images of lifeact-GFP distribution in non-stimulated Caco-2 cells. Basal plane (0 µm) shows randomly oriented lamellipodia, and medial plane (+5 µm) shows cortical actin enrichment at the cell junctions). (G) Representative time-lapse images of actin rearrangement in neighboring cells during apoptotic cell extrusion. Scale bar, 20 µm. Cells were stimulated and images were acquired as in Fig. 8, A–C. (H) Reconstructed side view (xz) of time-lapse series from B. Arrows indicate incomplete phagocytic cup formation during failed corpse engulfment. (I) Percentage of extruded cells over time. Cell death was induced by illumination as described previously in the co-cultures treated with the indicated inhibitors. The time of extrusion was determined by complete gap closure in the imaging plane, determined by the CellMask signal. Data are pooled from three (C) or two (D) independent experiments (total number of cell death events as indicated). Data in I are pooled from two independent experiments (total number of cell death events as indicated in Fig. 9 H. Mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Scale bars, 20 µm.

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