Figure 2.
Visualization of transport vesicles to apical membrane containing sphingomyelin. (A) A schematic diagram of the construct to visualize intracellular trafficking of sphingomyelin. SS means the preprotrypsin signal peptide (MSALLILALVGAAVAFPVD). (B) SS-GFP-Lys is expected to reside in the Golgi apparatus or the transport vesicles budding from TGN due to the binding to sphingomyelin synthesized and accumulated in the Golgi apparatus. When transport vesicles were fused with the plasma membrane, SS-GFP-Lys is released into the medium. (C) Total cell lysate of EpH4 cells stably expressing SS-GFP-Lys, GFP-Lys, and SS-GFP-LysWA were resolved by SDS-PAGE and immunoblotted with anti-GFP mAb and anti-alpha-tubulin mAb. (D) Total lipids were extracted from parental EpH4 cells and EpH4 cells expressing SS-GFP-Lys by Bligh and Dyer method. The ratio of the amount of sphingomyelin (mg/dl) to the total amount of phospholipids (mM) were plotted for both samples. N = 3 from independent experiments; error bar, SD; ns, not significant by Student’s t test. (E) EpH4 cells stably expressing SS-GFP-Lys, GFP-Lys, and SS-GFP-LysWA were fixed and stained with anti-GM130 mAb (red). Only SS-GFP-Lys co-localized with GM130. Scale bar, 10 μm. (F) EpH4 cells were treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Then, total lipids were extracted by Bligh and Dyer method and the amount of total phospholipids (mM) and sphingomyelin (mg/dl) were quantified. N = 3 from independent experiments; error bar, SD; *, P = 0.0162; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (G) EpH4 cells stably expressing both SS-GFP-Lys and mScarlet-Giantin were treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Scale bar, 5 μm. (H) The quantification of the cytoplasmic SS-GFP-Lys vesicles in cells treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Icytosol was calculated as (total area of SS-GFP-Lys − the area of SS-GFP-Lys that is colocalized with Giantin)/Total area of GFP. N = 3 from independent experiments; error bar, s.d.; ****, P < 0.0001; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. Source data are available for this figure: SourceData F2.

Visualization of transport vesicles to apical membrane containing sphingomyelin. (A) A schematic diagram of the construct to visualize intracellular trafficking of sphingomyelin. SS means the preprotrypsin signal peptide (MSALLILALVGAAVAFPVD). (B) SS-GFP-Lys is expected to reside in the Golgi apparatus or the transport vesicles budding from TGN due to the binding to sphingomyelin synthesized and accumulated in the Golgi apparatus. When transport vesicles were fused with the plasma membrane, SS-GFP-Lys is released into the medium. (C) Total cell lysate of EpH4 cells stably expressing SS-GFP-Lys, GFP-Lys, and SS-GFP-LysWA were resolved by SDS-PAGE and immunoblotted with anti-GFP mAb and anti-alpha-tubulin mAb. (D) Total lipids were extracted from parental EpH4 cells and EpH4 cells expressing SS-GFP-Lys by Bligh and Dyer method. The ratio of the amount of sphingomyelin (mg/dl) to the total amount of phospholipids (mM) were plotted for both samples. N = 3 from independent experiments; error bar, SD; ns, not significant by Student’s t test. (E) EpH4 cells stably expressing SS-GFP-Lys, GFP-Lys, and SS-GFP-LysWA were fixed and stained with anti-GM130 mAb (red). Only SS-GFP-Lys co-localized with GM130. Scale bar, 10 μm. (F) EpH4 cells were treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Then, total lipids were extracted by Bligh and Dyer method and the amount of total phospholipids (mM) and sphingomyelin (mg/dl) were quantified. N = 3 from independent experiments; error bar, SD; *, P = 0.0162; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. (G) EpH4 cells stably expressing both SS-GFP-Lys and mScarlet-Giantin were treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Scale bar, 5 μm. (H) The quantification of the cytoplasmic SS-GFP-Lys vesicles in cells treated with DMSO (control) or 40 µM HPA-12 or 50 μM Myriocin for 36 h. Icytosol was calculated as (total area of SS-GFP-Lys − the area of SS-GFP-Lys that is colocalized with Giantin)/Total area of GFP. N = 3 from independent experiments; error bar, s.d.; ****, P < 0.0001; and ns, not significant by one-way ANOVA with Tukey’s post-hoc test. Source data are available for this figure: SourceData F2.

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