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. 2011 Aug 20;60(9):2370–2378. doi: 10.2337/db10-0550

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© 2011 by the American Diabetes Association.

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FIG. 2. — The effect of DHA and cholesterol depletion on ASM expression in HREC. A: DHA and MCD pretreatment of HREC significantly decreased cholesterol content of the caveolar fractions compared with control. B: The inhibitory effect of DHA on ASM activity was not mimicked by MCD, and cholesterol replenishment did not restore ASM activity in DHA-treated HREC in whole-cell lysates. However, cholesterol depletion inhibited ASMase activity, and cholesterol replenishment reversed the inhibitory effect of DHA on ASM activity in the plasma membrane fraction. C: ASM protein expression in caveolar fractions was isolated from control and DHA-treated HREC. The results are means ± SD of three independent experiments performed in triplicate. *P < 0.01 compared with control. D: Decrease in ceramide-to-sphingomyelin ratio in caveolae from DHA-treated HREC compared with control. There was no change in ceramide-to-sphingomyelin ratio in total plasma membrane. DHA prevents TNF-α–induced increase in ceramide-to-sphingomyelin ratio in the caveolae. The results are means ± SD of three independent experiments performed in triplicate. *P < 0.05 compared with control. E and F: DHA or ASMase small interfering RNA (siRNA) treatment prevents IL-1β–induced increase in ICAM-1 expression in HREC. Random siRNA was used as control for ASM siRNA experiments. The results are means ± SD of at least three independent experiments performed in triplicate. *P < 0.05 compared with control; #P < 0.05 compared with IL-1β–stimulated HREC.