Fig. 6: S1P regulates human RBC glucose uptake, PP2A activity and GLUT1 phosphorylation and surface localization.
A Intracellular S1P levels and (B) corresponding S1P efflux in human RBC after incubation without or with 1 µM Sph for 30 min followed by exposure or not to 1% BSA for 30 min (n = 6 each). C Glucose uptake rate in human RBC treated as in (A) (n = 9). D PP2A activity of human RBC treated with 1 µM Sph or vehicle (n = 6 each). E Representative flow cytometry histograms of surface GLUT1 as measured by HRBDEGFP binding after incubation with 1 µM Sph or vehicle for 30 min. F Quantification of GLUT1 fluorescence from human RBC treated with 1 µM Sph or vehicle (n = 4). MFI (mean fluorescence intensity). G Western blotting for GLUT1 Serine 226 phosphorylation (n = 6), and (H) glucose uptake rate in human RBC treated or not with 1 µM sphingosine for 20 min before a five-minute stimulation or not with 5 nM TPA at 37 °C (n = 5). The ‘n’ in the (A–H) refers to samples from individual patients. Data are presented as mean ± sd and tested with stack matched one-way ANOVA followed by Tukey’s (A–C, G, H); paired two-tailed t test (D, F). p* < 0.05; p** < 0.01; p*** < 0.001; p**** < 0.0001.
