Fig 1. Loss of flotillins causes a reduction in cellular and tissue sphingosine-1-phosphate.

A. Quantitative lipid mass spectrometry using calibration with defined standards, of WT and flotillin knockout MEFs. 1x10⁶ cells were analysed in each sample.; Cer, Ceramide; SM, sphingomyelin; Sph, sphingosine; S1P, sphingosine-1-phosphate. Data from 4 biological replicates, each data point being derived from a separate experiment. Bars are mean. Statistical test is one-way ANOVA with Dunnett’s multiple comparisons test. B. S1P levels in mouse tissues and serum as shown, determined by quantitative lipid mass spectrometry. Bars are means and SD, each point represents a single sample from a different animal. Student’s t-test. C. Western blot to show reduction in flotillin 2 expression in cells treated with siRNAs against flotillin 1 and flotillin 2. Full scans of the blot are included in S3 Data File. D. siRNAs against flotillin 1 and flotillin 2 cause a reduction in S1P levels in HeLa cells as determined by quantitative lipid mass spectrometry. Data are normalised so that the mean of the control samples = 1, the data shown are from two separate experiments, in each experiment 3 biological replicates were analysed. Bars are mean and SD. Student’s t-test.