mTORC2 plays essential roles in the epithelial cyst formation by promoting apical transport of vesicles containing sphingomyelin and podocalyxin. (A) Representative immunofluorescence images showing development of apical lumen in wild-type MDCK cysts incubated with DMSO (Control), 40 µM HPA-12 or 1 µM Torin-1. After plating indicated time, cysts were fixed and stained with phalloidin (red), mAb against GP135/podocalyxin (green) and DAPI (blue). Scale bar, 5 μm. (B) Quantification of cysts with normal lumens or multiple lumens in cells treated with indicated inhibitors at 48 h. (Control, N = 490; HPA-12, N = 507; Torin-1, N = 510 cysts from five independent experiments). (C) EpH4 cells stably expressing GFP-podocalyxin were transfected with mScarlet only or mScarlet-tagged-Rab35 dominant active mutant (Q67L) expression vectors and treated with 250 nM Torin-1 or 3 µM Ku-0063794 for 24 h. Transfected cells are indicated by the white-dotted line. Scale bar, 10 μm.