Figure 5. Suppression of mTORC2 pathway... | Rockefeller University Press

Figure 5.

$Suppression of mTORC2 pathway reduces the amount of sphingomyelin in the apical membrane and impairs microvilli formation. (A) Wild type EpH4 cells and Rictor KD cells were treated with bSMase to degrade sphingomyelin at the apical membrane. Then, bSMase was washed out and cultured in a normal medium, and the recovery rate of sphingomyelin to the apical membrane of each cell was evaluated by the number of cells to which His-RFP-Lysenin binds. Scale bar, 20 μm. (B) The time-course change of number of cells stained with His-RFP-Lysenin after washing out bSMase in wild-type EpH4 cells and Rictor KD cells. N ≥ 3 independent experiments; error bar, SD; ****, P < 0.0001; ***, P < 0.0003; and ns, not significant by Student’s t test. (C) The apical membrane fractions of wild-type EpH4 cells and Rictor KD cells were isolated and the amount of sphingomyelin (mg/dl)/total phospholipid (mM) were quantified. N = 3 independent experiments; error bar, SD; *, P = 0.0327 by Student’s t test. (D) Wild-type EpH4 cells stably expressing podocalyxin-GFP were treated with either DMSO (control) or 250 nM Torin-1 for 24 h. Cells were stained with anti-GFP mAb and anti-E-cadherin mAb. Scale bar, 10 µm. (E) Scanning electron microscopy of wild-type EpH4 cells, bSMase treated wild-type EpH4 cells and Rictor KD cells. Scale bar, 5 µm. (F) EpH4 cells stably expressing SS-GFP-Lys were transfected with either mScarlet only or mScarlet-tagged-Rab35 dominant active mutant (Q67L) expression vectors and treated with DMSO (Control), 250 nM Torin-1 or 3 µM Ku-0063794 for 24 h. Transfected cells are indicated by the white-dotted line. Scale bar, 10 μm.$

Suppression of mTORC2 pathway reduces the amount of sphingomyelin in the apical membrane and impairs microvilli formation. (A) Wild type EpH4 cells and Rictor KD cells were treated with bSMase to degrade sphingomyelin at the apical membrane. Then, bSMase was washed out and cultured in a normal medium, and the recovery rate of sphingomyelin to the apical membrane of each cell was evaluated by the number of cells to which His-RFP-Lysenin binds. Scale bar, 20 μm. (B) The time-course change of number of cells stained with His-RFP-Lysenin after washing out bSMase in wild-type EpH4 cells and Rictor KD cells. N ≥ 3 independent experiments; error bar, SD; ****, P < 0.0001; ***, P < 0.0003; and ns, not significant by Student’s t test. (C) The apical membrane fractions of wild-type EpH4 cells and Rictor KD cells were isolated and the amount of sphingomyelin (mg/dl)/total phospholipid (mM) were quantified. N = 3 independent experiments; error bar, SD; *, P = 0.0327 by Student’s t test. (D) Wild-type EpH4 cells stably expressing podocalyxin-GFP were treated with either DMSO (control) or 250 nM Torin-1 for 24 h. Cells were stained with anti-GFP mAb and anti-E-cadherin mAb. Scale bar, 10 µm. (E) Scanning electron microscopy of wild-type EpH4 cells, bSMase treated wild-type EpH4 cells and Rictor KD cells. Scale bar, 5 µm. (F) EpH4 cells stably expressing SS-GFP-Lys were transfected with either mScarlet only or mScarlet-tagged-Rab35 dominant active mutant (Q67L) expression vectors and treated with DMSO (Control), 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.