Dihydrolanosterol

Hepatic cell line of murine origin was grown in a humidified atmosphere of 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA): F12-Ham’s medium (GE Healthcare Life Science, South Logan, UT, USA) enriched with fetal bovine serum and insulin/transferrin/selenium. When cells reached a confluence of 90–100%, medium was removed, cells were washed twice with phosphate buffered saline followed by addition of medium free of fetal bovine serum, insulin, transferrin and selenium. Cells were then incubated for 6 h with 200 µM squalene (Sigma-Merck, Darmstadt, Germany) or with 200 nM lanosterol, dihydrolanosterol, zymostenol or desmosterol (Avanti Polar lipids, Alabaster, AL, USA). Each condition was tested in six replicates. Media were removed, cells were washed twice with phosphate buffered saline then collected and total RNA was extracted using Tri-reagent solution (Ambion, Austin, TX, USA). DNA contaminants were removed by TURBO DNAse treatment using DNA removal kit (Ambion, Austin, TX, USA). squalene and sterol effects were investigated at mRNA level by RT-qPCR assays.

Deuterium labeled water (D₂O, 99.8% pure) was purchased from Sigma. Cholesterol, lanosterol, dihydrolanosterol, ff-MAS, t-MAS, zymosterol, desmosterol, 7-dehydroCholesterol, d₅-zymosterol, d₆-lanosterol, and d₆-sitosterol were obtained from Avanti Polar Lipids (Alabaster, AL). 25-hydroxyCholesterol (25-OHC) was obtained from Steraloids, Inc. (Newport, RI). Methyl-cyclodextrin (MCD) was obtained from Cyclodextrin Technologies (High Springs, FL). d₅-zymosterol, d₆-lanosterol were bound to MCD in a 1:10 and 1:20, respectively, stoichiometric ratio for solubilization as previously described (Brown et al., 2002 (link)).