Squalene Synthase Activity Assay.

Squalene synthase activity was assessed as described earlier (Amin et al., 1992). Briefly, each assay was in 1 ml assay buffer (50 mM phosphate buffer, pH 7.4, containing 10 mM MgCl₂) containing 0.5 mM NADPH, 12 µg human liver microsomes, and compound or vehicle (dimethylsulfoxide) alone in 16-mm ×100-mm glass screw-cap tubes. All components were allowed to equilibrate for 10 minutes at 37°C before the addition of [³H]-FPP (50 nM 0.045 Ci/mmol; PerkinElmer, Wokingham, UK) for a further 10 minutes at 37°C. The reaction was stopped by the addition of 1 ml 15% KOH dissolved in EtOH. Tubes were incubated at 65°C for 30 minutes and then 5 ml petroleum ether was added and shaken for 10 minutes. The lower aqueous phase was frozen and the upper organic phase was transferred to clean glass tubes containing 2 ml distilled water. Then, 1.5 ml upper organic phase was removed and counted with 3 ml scintillation liquid on a Tri-Carb 2800 TR Liquid Scintillation Analyzer (PerkinElmer).