Inhibition of apical cell extrusion mechanisms also affects entosis. (A–E) Representative examples of live cells recovered from the embryo medium of krt4:GFP;p63dsRed hai1a^hi2217 embryos at 48 hpf, and quantification of their frequency. Note the filopodia extending from the cell in A. In the complex cluster depicted in D, one basal cell is completely within a peridermal cell (arrow), while a second basal cell (arrowhead) sits in a lumen formed by the joined peridermal cells. (E) Representative image of a complex peridermal cell cluster lacking basal cells, recovered after overnight treatment with the Rho kinase inhibitor Rockout. (A) Maximum intensity projection; (B–E) single plane of a z-stack. n = 303 cells/clusters per condition. See also Fig. S3 and Video 2. (F) Quantification of the number of cells extruded per fish at 48 hpf after blockage of components of the Matriptase pathway. n = 60–88 embryos. (G–I) Representative live single-plane images of cell clusters stained with Lysotracker (white, arrow in G) to label lysosomal-acidified cytoplasms, either recovered from the medium after 16 h of incubation (G) or within the skin of hai1a^hi2217 embryos (H). (I) Quantification of Lysotracker-positive engulfed basal cells on skin surface and in incubation medium. n = 64 basal cells (medium) or 5 embryos (skin surface). (J) Quantification of cells extruded per fish at 48 hpf after 16 h treatment with indicated drugs known to interfere with apical cell extrusion and entosis. To avoid lethal effects of blebbistatin, extruded cells were counted after 6 h of treatment. n = 52–79 embryos. (K and L) Quantification of the proportion of recovered clusters from J containing basal cells (K), and of the average number of basal cells per cluster (L). n = 111–395 cells/clusters. For quantifications and statistical significances, see legend of Fig. 1. Scale bars, 10 µm.