Figure 3.
3D confocal microscopy and CLEM analysis of structural features of entosis in TRAIL-stimulated HCT116 cells. (A and B) 3D confocal microscopy imaging of β-catenin (red), Hoechst (blue), and LysoTracker (green) confirms complete cell internalization in control (A) and TRAIL-treated (B) cells. Representative confocal images of early-stage (top) and late-stage (bottom) entotic structures are shown. Orthogonal views through z-stacks of depicted areas are shown next to each image. Scale bars: 10 μm. (C–E) CLEM analysis of entotic ultrastructures in TRAIL-treated cells. Representative 3D confocal microscopy images of Hoechst (blue) and LysoTracker (green) staining of early (C), middle (D), and late (E) stages of entosis events are shown (left). Scale bar: 10 μm. TEM images of corresponding entosis events are shown (right). Two depicted areas (white and red squares) imaged in higher magnifications are shown. Green, magenta, and yellow pseudocolored areas indicate lysosomes, autophagosomes, and mitochondria, respectively. Blue pseudocolored areas indicate nuclei. Scale bars in TEM images from left to right: 10 μm, 2 μm, and 1 μm (C2) or 500 nm (D2 and E2). (F and G) Late-stage inner cells undergo cathepsin B–mediated lysosomal cell death. (F) Representative time-lapse images of LysoTracker staining (green) in HCT116 cells stably expressing Lamp1-mScarlet plasmid (red) showing an inner cell undergoing entotic cell death. Scale bar: 10 μm. (G) Representative 3D confocal microscopy images of fluorogenic cathepsin B substrate (red), DRAQ5 (blue), and LysoTracker (green) of early- and late-stage entotic cells are shown. Scale bars: 10 μm.

3D confocal microscopy and CLEM analysis of structural features of entosis in TRAIL-stimulated HCT116 cells. (A and B) 3D confocal microscopy imaging of β-catenin (red), Hoechst (blue), and LysoTracker (green) confirms complete cell internalization in control (A) and TRAIL-treated (B) cells. Representative confocal images of early-stage (top) and late-stage (bottom) entotic structures are shown. Orthogonal views through z-stacks of depicted areas are shown next to each image. Scale bars: 10 μm. (C–E) CLEM analysis of entotic ultrastructures in TRAIL-treated cells. Representative 3D confocal microscopy images of Hoechst (blue) and LysoTracker (green) staining of early (C), middle (D), and late (E) stages of entosis events are shown (left). Scale bar: 10 μm. TEM images of corresponding entosis events are shown (right). Two depicted areas (white and red squares) imaged in higher magnifications are shown. Green, magenta, and yellow pseudocolored areas indicate lysosomes, autophagosomes, and mitochondria, respectively. Blue pseudocolored areas indicate nuclei. Scale bars in TEM images from left to right: 10 μm, 2 μm, and 1 μm (C2) or 500 nm (D2 and E2). (F and G) Late-stage inner cells undergo cathepsin B–mediated lysosomal cell death. (F) Representative time-lapse images of LysoTracker staining (green) in HCT116 cells stably expressing Lamp1-mScarlet plasmid (red) showing an inner cell undergoing entotic cell death. Scale bar: 10 μm. (G) Representative 3D confocal microscopy images of fluorogenic cathepsin B substrate (red), DRAQ5 (blue), and LysoTracker (green) of early- and late-stage entotic cells are shown. Scale bars: 10 μm.

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