(A) SMS-KCNR cells were pre-treated with increasing concentrations of the dihydroceramide desaturase inhibitor C8-CPPC for 30 minutes and then 0.5 μM C12-dhCCPS was added for 6 h. Cells were collected after 6 h and the conversion to C12-CCPS was measured by LC/MS as described in “Experimental Procedures.” (B) Total endogenous levels of ceramides (Cer), dihydroceramides (dhCer), (C) dihydrosphingosine (dhSph), dihydrosphingosine-1- phosphate (dhSph-1P), sphingosine (Sph), sphinogosine-1-phosphate (Sph-1P), (D) Cer species, (E) and dhCer species were measured by LC/MS as described under “Experimental Procedures.” The sphingolipid levels were normalized to total lipid phosphate. The data presented are representative of the mean of 2 independent experiments, performed in duplicate ± S.D. The error bars represent the standard deviations, and when not seen, they are smaller than the thickness of the lines on the graphs.