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. Author manuscript; available in PMC: 2019 Apr 21.
Published in final edited form as: Cell Rep. 2019 Mar 19;26(12):3212–3220.e4. doi: 10.1016/j.celrep.2019.02.073

Figure 2. Entotic and Linker Cell Death, and Lobe Scission, Are Disrupted by Expression of a PIP2 Sensor.

Figure 2.

(A) Entotic death in MCF-7 cells is inhibited by GFP-PLCδ-PH expression. Graph shows the mean percentage of entotic cells from three independent biological repeats that underwent cell death over 24 h of time-lapse imaging. Error bars represent SEM. n = 35 total cells analyzed for GFP control, n = 36 for GFP-PLCδ-PH. Right images show representative entotic cell that did not undergo cell death and maintained a lobe (arrow) protruding from a host cell expressing GFP- PLCδ-PH (green). H2B-mCherry expression in cell nuclei is shown in red. **p = 0.04 (Student’s t test).

(B) Expression of PLCδ-PH::Venus in U cells inhibits linker cell cytoplasmic translocation of mCherry::UtrCH. Graph shows the cortical-to-cytoplasmic ratio of mCherry::UtrCH fluorescence (blue line, left y axis) in a linker cell from the time of engulfment marked by lobe formation. Red line shows the cortical actin ratio from control linker cell for comparison (from Figure 1D). Black line (right y axis) shows the distance of lobe separation from linker cell over time.

(C) High-level PLCδ-PH::Venus expression (>2-fold over background fluorescence intensity), but not low-level expression (<2-fold over background), in U cells inhibits linker cell death. Graph shows the percentage of linker cells that underwent cell death (determined by loss of cortical actin) within 45 min of lobe formation inside of U cells expressing GFP (n = 32) or low (n = 12) and high (n = 23) levels of PLCδ-PH::Venus. **p < 0.015; ***p < 0.001 (Fisher’s exact test).

(D) Representative images of a U cell expressing lin-48p::PLCδ-PH::Venus (green) with an engulfed mCherry::UtrCH-expressing linker cell 25 min (top) and 120 min (bottom) after lobe formation. Grayscale images show mCherry::UtrCH and PLCδ-PH::Venus fluorescence from the region indicated by the hatched box. Note that the linker cell maintains cortical actin as compared to the linker cell from a control experiment (right images, GFP-expressing U cell). See also Video S6.

(E) A surviving linker cell inside a PLCδ-PH::Venus-expressing U cell maintains an attached lobe. Images show linker cell mCherry::UtrCH fluorescence over the indicated time course. Note that the lobe (arrow, top images, maximum projections) stays attached, and cortical actin (bottom images, individual x-y plane images) is maintained. See also Video S7.

Scale bars, 10 μm.