FIGURE 3.

Effects of 4-HPR and its metabolites on in situ desaturase activity and endogenous sphingolipids. A, structures of 4-HPR and its metabolites. B, desaturase activity was measured using the in situ assay. SMS-KCNR cells were treated with increasing concentrations of 4-HPR, 4-oxo-4-HPR, 4-MPR, or 4-oxo-4-MPR for 6 h. C₁₂-dhCCPS was added at the same time as 4-HPR or its metabolites. Cells were collected after 6 h, and the conversion to C₁₂-CCPS was determined by LC/MS. Total endogenous levels of ceramides (Cer) and dihydroceramides (dhCer) in SMS-KCNR cells treated with 4-HPR (C), 4-oxo-4-HPR (D), 4-MPR (E), or 4-oxo-4-MPR (F) were measured by LC/MS as described under “Experimental Procedures.” Sphingolipid levels were normalized to total lipid phosphate. The data presented are representative of the mean of two independent experiments ± S.D. The error bars represent the S.D., and when not seen, they are smaller than the thickness of the lines on the graphs.