Downregulation of SK-1 results in sphingosine accumulation.

<p>(A) MDA-MB-231, NCI-H358, and HCT 116 cells were transduced with lentiviral SK-1 shRNA to downregulate SK-1 expression (SK-1 kd) or left untreated (wild-type, wt). MDA-MB-231 cells transduced with empty virus or transfected with the SK-1 cDNA for overexpression (SK-1 ov) are shown for comparison. Cell lysates were prepared and SK-1 protein expression was detected by Western blotting using antibodies against SK-1 (dilution 1∶1000) or GAPDH (dilution 1∶2000) as loading control. The two SK-1 splice variants SK-1a and SK-1b run at 43 and 51 kDa, respectively. The films were digitized and for each protein lane a density blot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells. (B) cellular sphingosine was determined in the genetically modified SK-1 kd carcinoma cell lines, in the respective wt cells, and additional MDA-MB-231 control cells as described above (empty virus transfected, SK-1 ov). Lipid extractions were performed and sphingosine was quantified by mass spectrometry. Data are means ± SD (<i>n = </i>3); *p<0.05, **p<0.01, ***p<0.001 compared to wt cells. (C) endogenous sphingosine in the wt carcinoma cell lines upon treatment with the SK-1 inhibitor SK I II (10 µM) or DMSO as vehicle control for 24 h. Quantification was done as above. Data are means ± SD (n = 3); *p<0.05, **p<0.01 compared to DMSO treated cells.