FIGURE 10.

Effect of SPHK1 and SPHK2 knockdown on l-OHP-induced ceramide formation, Akt phosphorylation, and pro-apoptotic protein expression in RKO cells. A, RKO cells were transfected with siRNA of SPHK1(1), siRNA of SPHK2(1), or nonspecific (NS) siRNA with Lipofectamine 2000 according to the manufacturer's instructions. After 24 h, the transfected cells were treated with or without l-OHP (5 μg/ml) for 24 h. The lipids were extracted from the cells, and the cellular ceramide concentration was measured by LC-MS/MS as described under ”Experimental Procedures.“ Results are expressed as percent increase of cells transfected with NS siRNA without l-OHP treatment, shown as means ± S.D. of three different experiments. *, p < 0.05. B, RKO cells transfected with siRNAs of SPHK1(1), SPHK2(1), or NS siRNA were treated with or without l-OHP (5 μg/ml). After 24 h, the cell lysates were subjected to Western blot analysis using the antibodies against phosphospecific-Akt, total Akt, p53, and p21. β-Actin was used as the loading control.