GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.A) HFS cells were treated with BFA (left panel) or monensin (right panel) with increasing concentrations for 24 hours. The GLTP mRNA expression levels were analyzed using qPCR and corrected to an 18S rRNA internal control. B) qPCR analysis of GLTP expression (filled circles) and sphingolipid levels in HSF cells treated with BFA (0.01 µg/ml, left panel) or monensin (5 µg/ml, right panel) for 6, 12 and 24 hours, 3H-sphinganine incorporation into the sphingolipids was analyzed using TLC. qPCR results are expressed as means +/− SD of at least three independent experiments. The data for the incorporation of the radiolabeled 3H-sphinganine are from at least three different experiments, and the results are normalized to the controls. Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls. C) Western blot analysis of GLTP levels in HSF cells treated with BFA (0.01 µg/ml) or monensin (5 µg/ml) for 24 hours. C = untreated control, B = BFA treatment, M = monensin treatment. β-Actin was used as a loading control. The representative blot shown here was chosen from one of three independent experiments with similar results.
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