Figure 1.
Single-cell time-lapse microscopy reveals simultaneous induction of entosis and apoptosis upon TRAIL stimulation. (A–D) HCT116 cells exhibit heterogeneity in caspase activation kinetics in response to TRAIL but not TRAIL + CHX. (A) Representative time-lapse images of HCT116 cells stably expressing IETD-FRET probe treated with TRAIL + CHX. Venus, TMRM, and CFP/FRET emission ratio images are shown. Depicted area highlights an apoptotic cell. Scale bar: 20 μm. (B) Schematic representation of the intact (left) and the cleaved (right) forms of IETD-FRET probe. Ex is the excitation wavelength; see Materials and Methods for details. (C) Quantification of single-cell CFP/FRET emission ratio traces in response to TRAIL (n = 57, left) and TRAIL + CHX (n = 60, right). (D) Single-cell IETD substrate cleavage kinetics in response to TRAIL and TRAIL + CHX. Data are shown as individual values for each cell as well as median and quartiles from three experiments. ****, P < 0.0001 by unpaired two-tailed t test. (E–H) Live cell TMRM staining accumulates in inner cells during entotic cell death. Representative time-lapse images of control (E and F) and TRAIL-treated (G and H) HCT116 IETD showing formation of entotic structures. DIC, Venus (green), TMRM (red), and CFP/FRET emission ratio images are shown. Representative entosis events are highlighted in a circle (entotic 1), a square (entotic 2), and a hexagon (entotic 3). Scale bar: 20 μm. Combined Venus (green) and TMRM (red) images highlighting stages of depicted entosis events (H). White-dashed and yellow-dashed lines indicate inner cells and outer cells, respectively. White asterisk indicates another entotic event in an outer cell of interest. Entotic 1, inner cell shows apoptotic features, then undergoes entotic cell death; entotic 2, inner cell shows caspase activation and loss of TMRM signal, then undergoes entotic cell death; entotic 3, inner cell with caspase activation invades into another cell, then is released. Scale bar: 20 μm. (I–K) Outer cells exhibit slow caspase activation kinetics and show less cleaved caspase-3 levels in TRAIL treatment. (I) Quantification of CFP/FRET emission ratio traces of inner (black) and outer (gray) cells in entotic 1, entotic 2, and entotic 3. Dashed line with arrow indicates time of internalization. Red dot on the single-cell trace of interest represents time of TMRM intensity loss. (J) Quantification of maximum mean values of CFP/FRET emission ratio traces in outer cells, apoptotic cells, or neighboring cells in HCT116 treated with or without TRAIL. Single-cell CFP/FRET emission ratio traces of n = 52 cells (13 cells/group) were generated over 20 h of time-lapse microscopy, and the maximum value in each trace was recorded. Data are shown as individual values for each cell as well as median and quartiles from three experiments. ****, P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. (K) Quantification of immunofluorescence staining of cleaved caspase-3 (Asp175) levels in apoptotic cells (n = 36) and outer cells (n = 30) in HCT116 treated with TRAIL. Entosis events were characterized using 3D confocal microscopy images of DIC and Hoechst by detecting a round-shaped Hoechst-stained cell inside a vacuolar structure within another Hoechst-stained cell showing a crescent-shaped nuclear morphology. Cells showing fragmented nuclei and increased cleaved caspase-3 intensity were considered apoptotic. Data are shown as individual values for each cell as well median and quartiles from three experiments. ****, P < 0.0001 by unpaired two-tailed t test. MFI, mean fluorescence intensity.

Single-cell time-lapse microscopy reveals simultaneous induction of entosis and apoptosis upon TRAIL stimulation. (A–D) HCT116 cells exhibit heterogeneity in caspase activation kinetics in response to TRAIL but not TRAIL + CHX. (A) Representative time-lapse images of HCT116 cells stably expressing IETD-FRET probe treated with TRAIL + CHX. Venus, TMRM, and CFP/FRET emission ratio images are shown. Depicted area highlights an apoptotic cell. Scale bar: 20 μm. (B) Schematic representation of the intact (left) and the cleaved (right) forms of IETD-FRET probe. Ex is the excitation wavelength; see Materials and Methods for details. (C) Quantification of single-cell CFP/FRET emission ratio traces in response to TRAIL (n = 57, left) and TRAIL + CHX (n = 60, right). (D) Single-cell IETD substrate cleavage kinetics in response to TRAIL and TRAIL + CHX. Data are shown as individual values for each cell as well as median and quartiles from three experiments. ****, P < 0.0001 by unpaired two-tailed t test. (E–H) Live cell TMRM staining accumulates in inner cells during entotic cell death. Representative time-lapse images of control (E and F) and TRAIL-treated (G and H) HCT116 IETD showing formation of entotic structures. DIC, Venus (green), TMRM (red), and CFP/FRET emission ratio images are shown. Representative entosis events are highlighted in a circle (entotic 1), a square (entotic 2), and a hexagon (entotic 3). Scale bar: 20 μm. Combined Venus (green) and TMRM (red) images highlighting stages of depicted entosis events (H). White-dashed and yellow-dashed lines indicate inner cells and outer cells, respectively. White asterisk indicates another entotic event in an outer cell of interest. Entotic 1, inner cell shows apoptotic features, then undergoes entotic cell death; entotic 2, inner cell shows caspase activation and loss of TMRM signal, then undergoes entotic cell death; entotic 3, inner cell with caspase activation invades into another cell, then is released. Scale bar: 20 μm. (I–K) Outer cells exhibit slow caspase activation kinetics and show less cleaved caspase-3 levels in TRAIL treatment. (I) Quantification of CFP/FRET emission ratio traces of inner (black) and outer (gray) cells in entotic 1, entotic 2, and entotic 3. Dashed line with arrow indicates time of internalization. Red dot on the single-cell trace of interest represents time of TMRM intensity loss. (J) Quantification of maximum mean values of CFP/FRET emission ratio traces in outer cells, apoptotic cells, or neighboring cells in HCT116 treated with or without TRAIL. Single-cell CFP/FRET emission ratio traces of n = 52 cells (13 cells/group) were generated over 20 h of time-lapse microscopy, and the maximum value in each trace was recorded. Data are shown as individual values for each cell as well as median and quartiles from three experiments. ****, P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. (K) Quantification of immunofluorescence staining of cleaved caspase-3 (Asp175) levels in apoptotic cells (n = 36) and outer cells (n = 30) in HCT116 treated with TRAIL. Entosis events were characterized using 3D confocal microscopy images of DIC and Hoechst by detecting a round-shaped Hoechst-stained cell inside a vacuolar structure within another Hoechst-stained cell showing a crescent-shaped nuclear morphology. Cells showing fragmented nuclei and increased cleaved caspase-3 intensity were considered apoptotic. Data are shown as individual values for each cell as well median and quartiles from three experiments. ****, P < 0.0001 by unpaired two-tailed t test. MFI, mean fluorescence intensity.

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