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

<p><b>A.</b> 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>B.</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. <b>C</b>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197401#pone.0197401.s003" target="_blank">S3 Data File</a>. <b>D</b>. 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.