Loss of mTORC2 activity did not affect biosynthesis of sphingomyelin or formation of transport vesicles of sphingomyelin from TGN (related to Fig. 4 ). (A) Wild-type EpH4 cells were treated with DMSO (Control), 250 nM Torin-1 (mTOR inhibitor) or 3 μM Ku-0063794 (mTOR inhibitor) for 72 h. Total cellular lipids were extracted by Bligh and Dyer method. The total amount of phospholipid (mM) and the amount of sphingomyelin (mg/dl) were quantified. N = 3 independent experiments; error bar, SD; *, P = 0.0140; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (B) Total phospholipids of wild type EpH4 cells (WT) and Rictor KD cells were extracted by Bligh and Dyer method. Total amount of phospholipids (mM) and the amount of sphingomyelin (mg/dl) were quantified. N = 3 from independent experiments; error bar, SD; ns, not significant by Student’s t test. (C) Wild-type EpH4 cells (WT) and Rictor KD cells (Rictor KD) co-expressing SS-GFP-Lys and mScarlet-mGiantin were cultured at 20°C for 2 h to block export from the TGN. This incubation resulted in depletion of cytoplasmic signals of SS-GFP-Lys and accumulation of SS-GFP-Lys in the Golgi apparatus. Release of the 20°C block by incubating the cells at 37°C for just 15 min resulted in the reappearance of SS-GFP-Lys containing vesicles in the cytoplasm. No significant difference was observed in the number of SS-GFP-Lys positive vesicles from Golgi bodies between wild-type EpH4 cells and Rictor KD cells. Scale bar, 5 μm.