Inhibition by LY294002 of IL-2-induced ceramide reduction and enzymatic changes in GCS, SMS, and acid and neutral SMases. KHYG-1 cells were cultured in the absence of IL-2 for 36 hours or rescued with IL-2 supplementation (100 U/mL, IL-2^-→+) and treated with various concentrations of LY294002 (0, 10, 20, and 30 μM) for 24 hours after 12 hours of deprivation. Ceramide content (A) was assessed by DGK assay, and the activities of GCS (B), SMS (C), acid (D), and neutral (E) SMases were measured by using C6-NBD-ceramide and C6-NBD-SM as the substrates as described in “Materials and methods.” Ceramide content after 24 hours of IL-2 supplementation was 4.6 ± 0.8 pmol/nmol phosphate. The activity of each enzyme detected after 24 hours of IL-2 supplementation was 789 ± 66, 41 ± 1.6, 579 ± 25, and 917 ± 91 pmol/mg protein/hour for GCS, SMS, acid, and neutral SMases, respectively. The bars indicate 1 SD. The significance of differences between the control () and LY294002-treated samples was determined by ANOVA test. ^*P < .01; ^**P < .05; #P > .05.