FIGURE 4.

Analysis of sphingosine and ceramidases. A, steady state intracellular levels of sphingosine in HT-GFP and HT-CerS6 cells normalized to inorganic phosphate. Data shown are the mean ± S.E. from three independent experiments. **, p < 0.005. B, qRT-PCR analysis of ceramidase mRNA. Data shown are from at least two independent determinations. C, Western blot analysis of acid ceramidase in SW620 and HT29 cells stably transfected with GFP or CerS6. Actin serves as loading control. D, acid ceramidase activity assay. Each assay was performed in triplicate. E, incorporation of ¹⁷C-sphingosine into sphingosine-1-phosphate. Data shown are the mean ± S.E. from two independent experiments. F, Western blot analysis of two individual clones of SW480 cells stably transfected with an inducible shRNA that targets CerS6. Protein lysates, generated after induction of shRNA expression for 1 week, were analyzed for CerS6 and acid ceramidase expression. Actin serves as loading control.