FIG 5.

Knockdown of MARCH6 increases SM activity. (A) SRD-1 cells were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and then treated with or without 20 μg/ml cholesterol-cyclodextrin (Chol/CD) for 8 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated. (B) SRD-1 cells were transfected with control or MARCH6 siRNA for 24 h, statin pretreated overnight, and subsequently incubated with [¹⁴C]acetate for 2 h. A statin-treated control was included to determine the background. Nonsaponifiable lipids were isolated and measured by scintillation counting. Data are means ± SEM from 3 independent experiments. (C) SRD-1 cells were transfected with control (Ctrl) or MARCH6 (M6) siRNA for 24 h and statin pretreated overnight, and total cell lysates were subjected to SDS-PAGE and immunoblotted as indicated (top panels) or incubated with [¹⁴C]mevalonate for 2 h in the presence of 10 μM lanosterol synthase inhibitor (bottom panels). Nonsaponifiable lipids were separated by thin-layer chromatography and visualized using phosphorimaging. MOS, monooxidosqualene; DOS, dioxidosqualene; Ctrl, control. SM activity is expressed as the DOS/MOS ratio with means ± SEM from 3 independent experiments. The control siRNA condition has been set to 1.