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Assayofdenovocholesterolsynthesis. On day 0, the cells were plated in 6-cm dishes in DMEM containing 10% FCS. On day 1, the medium was replaced with DMEM containing 5% LPDS, 4 μM lovastatin, and 100 μM mevalonate. On day 2, the cells were labeled with 0.5 μCi/ml [14C]acetic A-769662 for 2 h. Cells were then washed twice with PBS. Lipids were extracted and separated on TLC using hexane/diethyl ether/acetic acid (80:20:1) as a solvent. Incorporation of [14C]acetic acid into [14C]cholesterol was quantified by BAS 2000 (FUJIFILM).
Othermethods. Protein concentration was determined using the Bradford or BCA assays.
Results
Selective decrease of glycosphingolipids (GSLs) in GM95 cells
GM95 is a melanoma cell mutant which is defective in ceramide glucosysltransferase (GlcT-I) and thus does not synthesize GSL. MEB4 is the parent cell of GM95 whereas CG1 is a transfectant of GlcT-I in GM95. In Fig. 1, we measured lipid composition of MEB4, GM95, and CG1 cells after cells were labeled with [14C]serine. As previously reported [9], GM95 is deficient in GSLs, glucosylceramide, and GM3. Ceramide content was similar among the three cell lines. The content of [14C]serine-labeled phosphatidylethanolamine and phosphatidylserine was also similar among the three cell types. There was a significant increase of sphingomyelin in GM95 cells compared to MEB4 and CG1 cells as described [13].