EGF increases metabolic flux toward VLC-SM synthesis. (A) Incubation of cells with EGF results in increased VLC-SM synthesis. Cells were incubated with the indicated amounts of EGF (0.3–100 ng/ml) during the pacSph chase period. Product percentages for pacVLC- and pacLC-SM are described. (B) An EGFR kinase inhibitor, Gefitinib, inhibits cholesterol-dependent increase of VLC-SM synthesis. Cells were incubated with 10 μM Gefitinib overnight. pacSph pulse-chase experiments were performed and product percentages for pacVLC- and pacLC-SM are described. Statistical significance was evaluated by one-way ANOVA analysis and post hoc Dunnett’s (A) or Šídák’s (B) multiple comparisons were performed (P value >0.05, ns; <0.05, *; <0.01, **).