Lanosterol

cholesterol depletion media contained sterile-filtered (0.2 μm) 10 mM MβCD in 1× Dulbecco’s Modification of Eagle’s Medium (DMEM, Corning, 10–013-CV). Sterol replacement media contained 2.5 mM MβCD and a range of sterol concentrations (0.04 mM–0.6 mM), depending on the assay (unless indicated otherwise, a concentration of 0.2 mM sterol was used). To prepare sterol replacement media, various sterols in chloroform were added to sterile glass test tubes and dried under a stream of nitrogen gas. MβCD in 1 mL DMEM was added to the dried sterols. The solutions were mixed for 10 min in a shaking incubator at 250 rpm and 37 °C. After 10 min, solutions were sonicated in an ultrasonic bath for 1 min and mixed in a shaking incubator for 1–2 h at 37 °C before adding DMEM to the desired volume. The solutions were sterile filtered before use with the HEK 293T cells.
The following sterols were used for preparing the sterol replacement media: cholesterol from Avanti Polar Lipids; ergosterol, allocholesterol, dihydrocholesterol, and 7-dehydrocholesterol from Sigma Aldrich; 4-cholesten-3-one, coprostanol, desmosterol, epicholesterol, lathosterol, and lanosterol from Steraloids. The sterols generally appeared as single bands on thin layer chromatography, but lanosterol was purchased as a mixture with ~65% lanosterol, the remainder likely being dihydrolanosterol.

Lanosterol (55–65%; Steraloids, Newport, RI) was purified by silica gel chromatography using ethyl acetate:hexane (1:4) as a solvent. To Lanosterol (2 mmol, 0.85 g) in dichloromethane (40 mL) 5% Pd/C [20% (w/w), 0.17 g] was added and the mixture hydrogenated at room temperature at 50 psi for 24 h. The resulting mixture was filtered through celite, and the filter cake was washed thrice with 5 mL of dichloromethane. The solvent was evaporated to afford 24,25-dihydroLanosterol (DHL) as a white solid in quantitative yield. To improve the aqueous solubility, DHL was encapsulated in 2-hydroxypropyl-β-D-cyclodextrin (HPCD). To 9.5 mL of a stirring and refluxing solution of 45% (w/v) HPCD in 100 mM potassium phosphate buffer (pH 7.5), 2.1 mg of DHL was added and allowed to dissolve. After cooling to room temperature, the solution was diluted to 500 μM DHL. HPCD-encapsulated DHL (HPCD-DHL; DHL hereafter) was aliquoted and lyophilized for storage and reconstituted with deionized water prior to use.