FIGURE 4.

SMS1 overexpression attenuates the ability of cells to accumulate TG. A–C, HepG2-SMS1 and HepG2-EV cells were incubated with 1 mm palmitic acid delivered as a BSA complex (2:1, mol/mol) or with vehicle control (0.5 mm BSA) for 18 h. A, formation of lipid droplets visualized by Oil Red-O staining. B and C, levels of TG (B) and total cholesterol (C) measured in total lipid extracts as described under “Experimental Procedures.” Mean values ± S.D. (error bars) are shown (n = 3 dishes/point). D–F, HepG2 cells transiently transfected with SMS1 or EV were incubated with 0.1 or 1 mm ³H-labeled palmitic acid for 18 h. The specific labeling in each case was kept at 50 mCi/mmol. Lipids were extracted and separated by TLC as described under “Experimental Procedures.” D, radioactivity from the bands corresponding to SM quantified by scintillation counting. E, representative scan for ³H-labeled TG at 1 mm palmitic acid. F, radioactivity associated with TG determined by scintillation counting. According to two-way analysis of variance, the main effects of palmitate treatment and SMS1 overexpression on TG were statistically significant. The interaction effect was not statistically significant. The results of Bonferroni post-test analyses are indicated (***, p < 0.001; **, p < 0.01; *, p < 0.05). Results were confirmed in at least four independent experiments.