Analysis of 4-HPR-mediated alterations in dhSph and Sph phosphorylation. Externally added unnatural dhSph (17C-dhSph) and Sph (17C-Sph) analogues were used to estimate the secondary effects of 4-HPR-induced DES inhibition. CCRF-CEM cells were exposed to 4-HPR (10 μM) for 1 h and unnatural 17C species added for an additional 30 min incubation, as described in Methods. The effect on endogenous SLs (A) and unnatural dhSph species (B,C) analyzed by LC/MS. Comparative exogenous and endogenous SL profiles among parental (CCRF-CEM) and resistant cell lines (R0.5, R10) (D, E) were analyzed by the same methodology. Data are average ± SD of an experiment performed in duplicate.