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. 2021 Jan 23;296:100201. doi: 10.1074/jbc.RA120.012941

Figure 7.

Figure 7

TMR-S1P exhibits inhibitory properties to S1P release in erythrocytes. A, B, competition assays of sphingosine analogs with [3-3H]-Sph. TMR-Sph treatment showed an inhibitory effect on [3-3H]-S1P release in WT red blood cells (RBCs) (A), which concomitantly increased intracellular levels of S1P (B). WT and Mfsd2b KO RBCs were preincubated with 2 μM indicated sphingosine analogs for 2 h, then 2 μM [3-3H]-Sph and 0.5% BSA were added. The mixture was further incubated for 40 min. C, D, dose-dependent inhibition of [3-3H]-S1P release from TMR-Sph treatment. TMR-Sph treatment reduced the extracellular level of [3-3H]-S1P (C), which increased the intracellular [3-3H]-S1P (D) from WT RBC. WT and KO RBCs were preincubated with an indicated concentration of either unlabeled sphingosine or TMR-Sph for 2 h, then 1 μM [3-3H]-Sph and 0.5% bovine serum albumin were added. The mixture was further incubated for 30 min. Ethanol was used as vehicle as sphingosine was dissolved in ethanal. In comparison with unlabeled sphingosine, TMR-Sph shows inhibitory effects on the release of [3-3H]-S1P. n = 4 for each genotype. E, F, time course of TMR-Sph treatment. WT and KO RBCs were preincubated with 2 μM TMR-Sph for the indicated times. Then 1 μM [3-3H]-Sph and 0.5% bovine serum albumin were added. The mixture was further incubated for 30 min before S1P from cell pellets and supernatant were isolated and quantified. n = 4 for each genotype. G, TMR-Sph treatment does not reduce expression of Mfsd2b in erythrocytes. H, I, cellular thermal shift assay (CETSA) of wildtype erythrocytes with TMR-S1P. Erythrocytes were treated with 2 μM TMR-Sph or ethanol as vehicle for 1 h. Treated erythrocytes were heated at indicated temperatures. Mfsd2b protein expression was analyzed by Western blot and quantified. Mfsd2b protein from erythrocytes treated with TMR-Sph was significantly more stable at 55 and 60 °C. n = 6 for each genotype. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. p Values were calculated using one-way ANOVA. Data are expressed as mean and SD.