Figure 6.
Activation of Rab35 promotes apical transport of sphingomyelin via reduction of actin cortex of apical membrane. (A) EpH4 wild-type (WT) cells and Rictor KD cells stably expressing either mScarlet only or mScarlet-tagged-Rab35 Q67L were treated with bSMase. bSMase was washed out and cells were cultured in normal medium for an additional 24 h. Recovery of sphingomyelin at the apical membrane was imaged by recombinant GFP-Lysenin. Scale bar, 10 μm. (B) Time course change in the number of GFP-Lysenin-positive cells at the apical membrane was quantified. N ≥ 3 independent experiments; error bar, SD. (C) EpH4 wild-type (WT) cells and Rictor KD cells were stained with phalloidin. Scale bar, 10 μm. (D) Quantification of the fluorescent intensity of phalloidin at the apical and basolateral membrane in wild-type EpH4 cells (black) and Rictor KD cells (red). N = 5 from independent experiments; error bar, SD, two-way ANOVA; *, P = 0.0476; ****, P < 0.0001. (E) EpH4 wild-type (WT) cells and Rictor KD cells stably expressing either mScarlet only or mScarlet-Rab35 Q67L were stained with phalloidin. Scale bar, 10 μm. (F) Quantification of the fluorescence intensities of phalloidin at the apical membrane in mScarlet only expressing Rictor KD cells and mScarlet-Rab35 Q67L expressing Rictor KD cells. N = 10 independent experiments; error bar, SD; **, P = 0.0048 by Student’s t.test.

Activation of Rab35 promotes apical transport of sphingomyelin via reduction of actin cortex of apical membrane. (A) EpH4 wild-type (WT) cells and Rictor KD cells stably expressing either mScarlet only or mScarlet-tagged-Rab35 Q67L were treated with bSMase. bSMase was washed out and cells were cultured in normal medium for an additional 24 h. Recovery of sphingomyelin at the apical membrane was imaged by recombinant GFP-Lysenin. Scale bar, 10 μm. (B) Time course change in the number of GFP-Lysenin-positive cells at the apical membrane was quantified. N ≥ 3 independent experiments; error bar, SD. (C) EpH4 wild-type (WT) cells and Rictor KD cells were stained with phalloidin. Scale bar, 10 μm. (D) Quantification of the fluorescent intensity of phalloidin at the apical and basolateral membrane in wild-type EpH4 cells (black) and Rictor KD cells (red). N = 5 from independent experiments; error bar, SD, two-way ANOVA; *, P = 0.0476; ****, P < 0.0001. (E) EpH4 wild-type (WT) cells and Rictor KD cells stably expressing either mScarlet only or mScarlet-Rab35 Q67L were stained with phalloidin. Scale bar, 10 μm. (F) Quantification of the fluorescence intensities of phalloidin at the apical membrane in mScarlet only expressing Rictor KD cells and mScarlet-Rab35 Q67L expressing Rictor KD cells. N = 10 independent experiments; error bar, SD; **, P = 0.0048 by Student’s t.test.

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