Figure 4.

Engulfment and degradation of corpses provides a survival advantage

(A) Entotic death rate of internalized cells increases after exposure to UV radiation. Graph shows internalized cell death rates for MCF7 cells with (red) or without (black) exposure to UV. Death rates are determined over a 10 hr period after the completion of entosis, as determined by a vacuole appearance in the outer cell by DIC microscopy. Error bars depict mean ± SD. Data are from at least three independent experiments with at least 20 entotic pairs counted in each experiment.

(B) Entotic host cells survive at higher rates than neighboring single cells, in a lysosome-dependent manner. Graph shows the quantification of survival rates for entotic host cells and neighboring single cells after exposure to UV radiation with or without ConA treatment. Cell survival rates are determined by morphology using DIC microscopy. Error bars depict mean ± SD, with at least three independent experiments. Entotic host: n = 217; neighboring single cells: n = 312; entotic host with ConA: n = 167; neighboring single cells with conA: n = 435.

(C) Representative images depicting internalized cell degradation in MCF7 cells. The top panel shows internalized cell in UV-irradiated culture that becomes killed and digested over time; bottom panel shows failure of internalized cell degradation in ConA-treated culture, and host cell undergoes cell death (right, arrow). Blue dotted lines depict internalized cells.

(D) Phagocytosis of apoptotic corpses by J774.1 cells provides survival advantage. Graph shows quantification of cell survival in the presence or absence of apoptotic corpses and ConA. Image shows phagocytic ingestion of apoptotic corpse by J774.1 cell; blue dotted line shows corpse inside of phagosome. Error bars depict mean ± SD. Data are from three independent experiments with at least 1000 cells counted in each condition.

(E) Overall cell death rates determined by time-lapse microscopy after exposure to UV radiation in MCF7 and BxPC3 cells, in the presence or absence of Y27632 or Z-VAD-FMK. Y27632: ROCK inhibitor; Z-VAD-FMK: pan-caspase inhibitor. Error bars depict mean ± SD, with at least 350 cells quantified in each condition from three independent experiments. Statistics are obtained using Student's t-test.

(F) Frequencies of individual death types in MCF7 and BxPC3 cells exposed to UV radiation, determined by DIC morphology; representative images of entosis, apoptosis and necrosis shown on right. Note that the inhibition of entosis (with Y27632) results in more necrosis in MCF7 cells, while inhibition of apoptosis (with Z-VAD-FMK) results in more entosis in BxPC3 cells. Graph shows quantification of apoptosis, entosis, and necrosis after exposure to UV radiation, with the indicated treatment, as determined by time-lapse microscopy. Error bars depict mean ± SD, with at least 150 cells quantified in each condition from three independent experiments. Statistical tests were performed using Student's t-test.