Figure 4.
Inhibition of the mTORC2 pathway impairs apical transport of sphingomyelin. (A) EpH4 cells stably expressing SS-GFP-Lys and mScarlet-Giantin were treated with DMSO (Control), 250 nM Torin-1, or 3 μM Ku-0063794 for 12 h. Treatment with mTOR inhibitors increases the dot-like cytoplasmic SS-GFP-Lys vesicles without changing morphology of the Golgi apparatus. Scale bar, 10 μm. (B) Quantification of the cytoplasmic SS-GFP-Lys vesicles in cells treated with DMSO (Control), 250 nM Torin-1 or 3 μM Ku-0063794 for 24 h. N = 3 independent experiments; error bar, SD; **, P(Torin-1) = 0.0029; **, P(Ku-0063794) = 0.0052; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (C) Quantification of Pearson’s correlation coefficient between SS-GFP-Lys and Giantin in cells treated with DMSO (Control), 250 nM Torin-1, or 3 μM Ku-0063794 for 12 h. N = 7 from independent experiments; error bar, SD; ***, P = 0.0002; *, P = 0.0157; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (D) Total cell lysates of wild-type EpH4 cells (WT), Raptor knockdown (KD) and Rictor KD EpH4 cells were resolved by SDS-PAGE and immunoblotted with indicated antibodies. In Rictor KD cells, the level of Akt (Ser 473) phosphorylation are reduced as compared to wild-type EpH4 cells as described previously (Guertin et al., 2006). (E) Raptor or Rictor was knocked down in EpH4 cells stably expressing SS-GFP-Lys and mScarlet-Giantin. The cytoplasmic dots of SS-GFP-Lys were significantly increased in Rictor KD cells. Scale bar, 10 μm. (F) Quantification of the cytoplasmic SS-GFP-Lys vesicles in WT cells, Raptor KD cells and Rictor KD cells. (N = 8 from independent experiments; error bar, SD, one-way ANOVA; ***, P = 0.0003; ****, P < 0.0001). (G) Quantification of Pearson’s correlation coefficient between SS-GFP-Lys and Giantin in WT cells, Raptor KD cells and Rictor KD cells. N = 8 independent experiments; error bar, SD; *, P(WT) = 0.0243; *, P(Raptor KD) = 0.0232; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. Source data are available for this figure: SourceData F4.

Inhibition of the mTORC2 pathway impairs apical transport of sphingomyelin. (A) EpH4 cells stably expressing SS-GFP-Lys and mScarlet-Giantin were treated with DMSO (Control), 250 nM Torin-1, or 3 μM Ku-0063794 for 12 h. Treatment with mTOR inhibitors increases the dot-like cytoplasmic SS-GFP-Lys vesicles without changing morphology of the Golgi apparatus. Scale bar, 10 μm. (B) Quantification of the cytoplasmic SS-GFP-Lys vesicles in cells treated with DMSO (Control), 250 nM Torin-1 or 3 μM Ku-0063794 for 24 h. N = 3 independent experiments; error bar, SD; **, P(Torin-1) = 0.0029; **, P(Ku-0063794) = 0.0052; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (C) Quantification of Pearson’s correlation coefficient between SS-GFP-Lys and Giantin in cells treated with DMSO (Control), 250 nM Torin-1, or 3 μM Ku-0063794 for 12 h. N = 7 from independent experiments; error bar, SD; ***, P = 0.0002; *, P = 0.0157; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (D) Total cell lysates of wild-type EpH4 cells (WT), Raptor knockdown (KD) and Rictor KD EpH4 cells were resolved by SDS-PAGE and immunoblotted with indicated antibodies. In Rictor KD cells, the level of Akt (Ser 473) phosphorylation are reduced as compared to wild-type EpH4 cells as described previously (Guertin et al., 2006). (E) Raptor or Rictor was knocked down in EpH4 cells stably expressing SS-GFP-Lys and mScarlet-Giantin. The cytoplasmic dots of SS-GFP-Lys were significantly increased in Rictor KD cells. Scale bar, 10 μm. (F) Quantification of the cytoplasmic SS-GFP-Lys vesicles in WT cells, Raptor KD cells and Rictor KD cells. (N = 8 from independent experiments; error bar, SD, one-way ANOVA; ***, P = 0.0003; ****, P < 0.0001). (G) Quantification of Pearson’s correlation coefficient between SS-GFP-Lys and Giantin in WT cells, Raptor KD cells and Rictor KD cells. N = 8 independent experiments; error bar, SD; *, P(WT) = 0.0243; *, P(Raptor KD) = 0.0232; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. Source data are available for this figure: SourceData F4.

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