FIGURE 1.

Characterization of SMS1 protein expression and activity in HepG2 cells. The HepG2-SMS1 cell line stably overexpresses the human V5-tagged SMS1 (HepG2-SMS1), whereas HepG2-EV is the empty vector control cell line. A, expression and subcellular localization of the V5-tagged SMS1 protein (green) visualized by indirect immunofluorescence in permeabilized cells using antibody against the V5 tag. Hoechst 33258 (blue) and wheat germ agglutinin (WGA; red) were used for staining of the nuclei and Golgi. B, SM synthase activity measured in vitro. C–E, in situ labeling of SM in live cells using NBD-ceramide (C), [³H]palmitic acid (D), and BODIPY®-palmitic acid (E) as tracers. F, mass of SM measured by TLC separation of total lipid extract and quantification of inorganic phosphate. Mean values ± S.D. (error bars) are shown (n = 3 dishes/point). Results were confirmed in at least three independent experiments, and representative data are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 according to Student's t test.