Figure 6.
Genz treatment of Lovo and HCT116 cells lead to a concentration-dependent increase of sphingomyelin (SM). Lipids were quantified by C18-reversed phase UPLC/MS2 in MRM mode using internal standards for each lipid class. Major sphingolipids containing non-hydroxy fatty acids (N) and (C18)-sphingosine (S) were recorded; Cer, ceramide, HexCer: hexosylceramide, SM, sphingomyelin, and PC: phosphatidylcholine. (A,B) HexCer was reduced to a similar extent in Genz- or UGCG-gRNA-treated Lovo (A) or HCT116 cells (B). The SM content in Lovo (A) and HCT116 (B) cells was remarkably elevated upon Genz treatment and much higher as in cells treated with UGCG-gRNA in which the SM-increase correlated vice versa with the reduction of GSLs. The ceramide levels in Lovo and HCT116 cells upon Genz treatment were similar as in controls. (C,D) SM levels in Miglustat-treated Lovo (C) and HCT116 cells (D) were also elevated as compared to controls. Ceramides appeared unaltered upon Miglustat treatment; each data point on the graphs represents one biological replicate; significances are *, p ≤ 0.05; **, p < 0.01; ***, p < 0.001.