Fig. 3. Sphingolipid analysis of lysosome-targeted caged sphingosine. (A) Schematic illustration of the interconversion between sphingosine, sphingosine 1-phosphate (S1P) and ceramides. Sphingosine can be phosphorylated into S1P by sphingosine kinases (SphKs); and S1P can be dephosphorylated into sphingosine by sphingosine 1-phosphate phosphatases (SGPPs). Ceramide synthases (CerS 1–6) convert sphingosine into ceramides with various acyl chain lengths; ceramidases (CDases) cleave ceramides to yield sphingosine and fatty acids. (B) Analysis of D₇-sphingosine and D₇-S1P levels under various experimental conditions as indicated. (C) Changes of sphingosine and S1P levels over time after uncaging of D₇-Lyso-So in HeLa cells. The decay curves were generated by fitting into a single phase decay function. Data in (B) and (C) were normalized with respect to the amount of C17 internal standards and cell numbers. (D and E) Metabolism of newly formed D₇-ceramides after photo-releasing D₇-sphingosine from Lyso-So in HeLa cells. Data in (D) and (E) were normalized by the sum of all measured lipid signals and presented as “mol% of total”. All data represent the average of three independent experiments. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant, Students' t-test.