Dynamics of the apicosome in cell aggregates. (A) Schematic of the hPSC-aggregate lumen formation assay (see the legend to Fig. S3 K for a detailed description). (B–E) hESC aggregates at 0 h, stained with apical markers (PODXL [B], aPKCζ [B], p-ERM [C and D], and EZRIN [E]) in addition to markers of adherence junction (E-CADHERIN [C]), recycling endosome (RAB11 [D]), and primary cilia (ARL13B [E]). Dotted circles in green channel outline radially patterned cells. In E, the boxed regions indicate clustered EZRIN⁺ apicosomes in the center of radially patterned cells (i) and an EZRIN⁺ lumen studded with two primary cilia (ii). (F) Time-lapse imaging of H9 PODXL–GFP aggregates. Imaging began immediately after ROCK-i withdrawal (0 h), and the first image is time stamped (00:00). Time stamp, h:min. Bars: (B–F) 50 µm; (E-i) 5 µm; (E-ii) 2 µm. For all images, blue indicates DNA staining (HOECHST).