(A) Kinetics of ceramide generation induced by Dex treatment. Thymocytes were treated with Dex (10−7 mol/L) for different times. Lipids were then extracted, subjected to DAG kinase assay, and separated by TLC. Radioactive spots were visualized by autoradiography, scraped from the plate, and counted by scintillation counting. On the left, the quantitative results for ceramide-1-phosphate levels, expressed as picomoles/106cells are reported (mean values of three different experiments in duplicate; standard deviations, less than 10% of the mean values, are omitted for clarity). On the right side, a representative autoradiography of a ceramide chromatogram is shown. (B) C2-ceramide–induced apoptosis. Thymocytes were incubated for 18 hours with 10 μmol/L C2-ceramide in the presence or absence of actinomycin-D (Act-D; 2.5 μg/mL) and cycloheximide (CHX; 50 μg/mL) and analyzed for apoptosis. Percentage numbers of hypodiploid nuclei are reported for each condition. Data shown are representative of one out of two experiments. (C) Dex-induced SMase activation. Acidic (on the left) and neutral (on the right) SMase activity induced by Dex treatment at the indicated times. Hydrolyzed SM was quantitated and expressed as picomoles/106 cells. The mean values from two determinations are reported. Standard deviation values were lower than 3% of mean values. The results are representative of one out of three separate experiments.
