Figure 6.

FTY720 affects LAT1 localization. (A) Cells of the stable T-REx HeLa line expressing LAT1-GFP were transfected with a plasmid expressing Rab5-mCherry, grown for 24 h in the presence of tetracycline, and then for an additional 40 h in tetracycline-free medium. Live cell images were acquired after 10 min of treatment with FTY720 (5 μM final concentration) or DMSO solvent alone (control). The graph corresponds to quantification of the images with Pearson’s correlation coefficient. In the boxplots, the middle line denotes the median and the top and bottom of the box indicate, respectively, the 75th and the 25th percentile. The whiskers denote the maximum and minimum values. Unpaired t-test ***P ≤ 0.001, n ≥ 32 cells. (B) Cell-surface biotinylation assay for monitoring FTY720-induced LAT1-GFP endocytosis. Cells of the stable T-REx HeLa line expressing LAT1-GFP were grown as in A and incubated with sulfo-NHS-biotin for 30 min at 4 °C. The cells were then incubated in the presence of FTY720 (5 µM final concentration) or the DMSO solvent alone (control) for the indicated times at either 4 °C to block membrane trafficking (control reduction) or 37 °C. The cells were washed and the remaining surface biotin was then cleaved except in a control sample (total surface). Biotinylated proteins were purified by affinity chromatography with streptavidin-coated beads. LAT1-GFP was then detected by immunoblotting with anti-GFP antibody. A blot representative of three independent experiments is shown. (C) Cells of the stable T-REx HeLa line expressing LAT1-GFP were grown as in A. They were then treated for 6 h with DMSO (control) or 5 μM FTY720. The cells were fixed and the localization of LAT1-GFP and LAMP1 was examined by confocal microscopy. Nuclei were stained with DAPI.