Figure 2.

Inhibition of SphK1 reduces sphingosine uptake in erythrocytes. A, distribution of NBD-S1P in WT and KO red blood cells after incubation of NBD-sphingosine for 5 and 30 min. We interpreted the green signals as NBD-S1P as we have observed that the NBD-Sph level is low in red blood cells (see Results in Vu et al. [14]). However, we cannot discriminate NBD-S1P and NBD-Sph signals by microscopic assays. Representative images from three WT and three KO mice. Similar results were obtained for TMR-Sph. Arrowheads and arrows show NBD-S1P signals on the plasma membrane and in the cytoplasm, respectively. B, C, quantification of numbers of puncta in WT and Mfsd2b KO erythrocytes after incubation with NBD-Sph or TMR-Sph, respectively. n = 3 for each genotype. D, E, S1P levels in supernatant and cell pellets with or without treatment of 20 and 60 μM PF543. Radioactive S1P was isolated from the supernatant and cell pellets for quantification. S1P signals from WT without treatment were set as 100%. F, Sphingosine levels in the supernatant with or without 20 and 60 μM PF543. In these experiments, 2 μM [3-³H]-Sph was used in “continuous” assays for 15 min. Sphingosine signals from WT without treatment were set as 100%. Data are mean and SD, n = 4 per genotype. Experiments were repeated twice. ∗∗∗p < 0.001. p Values were calculated using one-way ANOVA.