Epicholesterol

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.