FIGURE 2:

Fate of internalized cells and host cells. (A) Representative images of Venus-Lgl2 cells from a live-cell, time-lapse analysis. Live cells were stained with Hoechst 33342 (blue). DIC and merged images from green and blue channels are presented. Time points are presented as hours:minutes. (B) TEM images showing degrading internalized Venus-Lgl2 cells. (C) A number of internalized cells die through apoptosis. Venus-Lgl2–expressing cells were fixed and stained with anti-cleaved caspase3 antibody (red). DNA was stained with Hoechst 33342. White and red arrowheads indicate dying internalized cells that are negative or positive for cleaved caspase3 staining, respectively. Scale bars: 10 μm. (D) Quantitation of internalized cells that exhibited nuclear disintegration and were cleaved-caspase3-positive in Venus-Lgl2–expressing cells at 16 h after plating. Cells were treated with dimethyl sulfoxide (DMSO; control), caspase inhibitor ZVAD-FMK (ZVAD, 20 μM), or autophagic/lysosomal inhibitor 3-MA (5 mM). Data were from three independent experiments. Error bars indicate SD values. (E and F) Venus-Lgl2–induced cell–cell internalization led to cytokinesis defect in the host cells. (E) Images of Venus-Lgl2 cells fixed at 12 h after plating. DNA was stained with Hoechst 33342. H, host cell; I, internalized cell. (F) Quantification of binucleated host cells at different time points after Venus-Lgl2 cells were plated on coverglass.