Fig. 2.

Sphingolipids inhibit recycling of nutrient transporters in a PP2A-dependent manner. (A) Loss of surface CD98 in FL5.12 and SupB15 cells. Cells were incubated with a primary unconjugated CD98 antibody on ice for 30 min. Cells were then treated with vehicle or FTY720 at 37°C for the indicated times, incubated on ice with secondary antibody for 30 min, and surface CD98 measured by flow cytometry. (B) Transferrin uptake in FL5.12 and SupB15 cells with or without FTY720. (C) CD98 recycling in SupB15 cells. Cells were incubated with primary CD98 antibody as in A, then treated with vehicle or FTY720 for 1 h at which point cells were washed and returned to vehicle- or FTY720-containing medium for 30 min. Surface CD98 levels were then measured by flow cytometry. (D) Tf recycling assay conducted in the presence or absence of FTY720. (E) FL5.12 cells pre-treated (1.5 h) with PP1 inhibitor tautomycin (TT, 200 nM) or PP2A inhibitors cantharidin (CT, 10 μM) or calyculin A (calA, 5 nM) were treated with FTY720 for 1 h and surface CD98 quantified by flow cytometry. (F) Internalization of surface CD98 was measured as in A, cells were pre-treated with calyculin A (5 nM) for 1 h where indicated. (G) Following surface labeling with a primary antibody, FL5.12 cells were treated with FTY720 for 1 h at which point vehicle or calyculin A (10 nM) was added for an additional 1 h. Cells were then stained with secondary antibody and surface CD98 measured using flow cytometry. FTY720 was used at 5 μM. Means±s.e.m. are shown; n≥3 in all panels. Two-tailed t-test was used to compare sphingolipid-treated time points with vehicle time points. n.s., not significant; *P≤0.05; ***P≤0.001.