Apoptotic cells produce and transmit S1P during extrusion. (A–C) Confocal fluorescence images during early (A), middle (B), and late (C) stages of extrusion of apoptotic cells from an HBE monolayer. (D and E) Confocal fluorescence images of blocked apoptotic cell extrusion by SKI II (D) or the S1P2 antagonist JTE-013 (E). Each experimental sample was visualized with five (B and C) or three (A, D, and E) consecutive 3D projections (comprising 2-µm thickness each), as necessary to span the full distance from the most basal to the most apical section (second-to-bottom and top images, respectively). Note that total cell height under the different conditions varies: during early extrusion (A) and when extrusion is blocked with SKI II and JTE-013 (D and E), the dying cell is not squeezed out of the epithelium and therefore does not inhabit as great an apical-to-basal distance as when the dying cell is extruding (B and C). A, B, C, and E were obtained using a confocal microscope (TCS SP5; Leica), whereas D was taken using an inverted microscope (Eclipse TE300; Nikon) converted for spinning disc confocal microscopy. A′–E′ represent zoomed-in region (square) from each montage. (D′′) Inset denoting that the unextruded cell in D is apoptotic. Bars, 10 µm.
