Apoptotic cell extrusion requires the S1P₂ receptor. (A) HBE cells induced to undergo apoptosis with UV in the presence of DMSO or the indicated S1P receptor antagonists. (B) qRT-PCR confirms shRNA-mediated knockdown of S1P₂ in HBE cells. (C) Quantification of nonextruded apoptotic cells in HBE monolayers expressing control or S1P₂-specific shRNA after UV treatment. (D and E) A dying HBE cell is not extruded by S1P₂-silenced cells (D, green), but is extruded successfully by normal surrounding cells (E). When S1P₂ shRNA is only in the dying cell (E), it extrudes and is in a higher plane than the actin ring below it, but is in the same plane when the surrounding cells are knocked down for S1P₂ (D). Projections of extruding and nonextruding apoptotic cells from WT (F) or mil (G) zebrafish larvae, respectively. (H and I) Cross sections (XZs) of an apoptotic extruding cell (H) and a nonextruding cell (I) from WT (H) and mil zebrafish larvae (I), respectively. For all bar graphs, each bar represents the average percentage of nonextruded apoptotic cells to total apoptotic cells with each treatment from three independent experiments; n = 100 dying cells per experiment, error bars = SDs. **, P < 0.01; ***, P < 0.001. Bars, 10 µm.