Supplementary video file for Methods and Protocols

Sphingolipid metabolism is an important process in sustaining the growth needs of rapidly dividing cancer cells. Enzymes that synthesize sphingolipids have become attractive targets in cancer pharmacology. Ceramide is a precursor for synthesizing sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and glucosylceramide. Sphingomyelin synthase (SMS) is the enzyme that transfers a phosphatidylcholine to ceramide to generate sphingomyelin. To test the inhibition of SMS, scientists assess the buildup of ceramide in the cell, which is cytotoxic. Because ceramide is a small lipid molecule there are limited tools like antibodies to detect its presence. Alternatively, designated machines for small molecule separation coupled with mass spectrometry detection can be used, but can be cost-prohibitive. We used a commercially available NBD-ceramide to apply to human cancer cell lines in the presence or absence of a known SMS inhibitor, jaspine B. After short incubation times, we were able to collect cell lysates and using solvent extraction methods, run the cellular material on a thin layer chromatography plate to determine the levels of intact fluorescently-labeled ceramide. The brighter the fluorescence on the TLC correlated to greater SMS inhibition. Small molecules can then be screened quantifiably to determine the biological impact of inhibiting the sphingolipid metabolism pathways involving ceramide.Sphingolipid metabolism is an important process in sustaining the growth needs of rapidly dividing cancer cells. Enzymes that synthesize sphingolipids have become attractive targets in cancer pharmacology. Ceramide is a precursor for synthesizing sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and glucosylceramide. Sphingomyelin synthase (SMS) is the enzyme that transfers a phosphatidylcholine to ceramide to generate sphingomyelin. To test the inhibition of SMS, scientists assess the buildup of ceramide in the cell, which is cytotoxic. Because ceramide is a small lipid molecule there are limited tools like antibodies to detect its presence. Alternatively, designated machines for small molecule separation coupled with mass spectrometry detection can be used, but can be cost-prohibitive. We used a commercially available NBD-ceramide to apply to human cancer cell lines in the presence or absence of a known SMS inhibitor, jaspine B. After short incubation times, we were able to collect cell lysates and using solvent extraction methods, run the cellular material on a thin layer chromatography plate to determine the levels of intact fluorescently-labeled ceramide. The brighter the fluorescence on the TLC correlated to greater SMS inhibition. Small molecules can then be screened quantifiably to determine the biological impact of inhibiting the sphingolipid metabolism pathways involving ceramide.