Dolichol

The culture media RPMI 1640, HEPES, gentamycin, and Albumax I were obtained from GIBCO Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). Glucose, sodium bicarbonate, and hypoxanthine were purchased from Millipore-Sigma (Burlington, MA, USA). Polyprenol, dolichol, polyprenal, and nor-dolichol mixtures of 13 to 21 isoprene units were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The dolichyl phosphate mixture (14 to 18 isoprene units), all trans-polyprenol (9 isoprene units), and isopentenol were obtained from Isoprenoids LC (Tampa, FL, USA). The following chemicals were obtained from Cayman Chemicals (Ann Arbor, MI, USA): polyunsaturated fatty acid mixture (adrenic acid, arachidonic acid, dihomo-γ-linolenic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, stearidonic acid), saturated and monounsaturated fatty acid mixture (arachidic acid, lauric acid, lignoceric acid, myristic acid, nervonic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid), and ubiquinone-10. Squalene, 2,3-oxidosqualene, cholesterol, β-carotene, menaquinone-4, α-tocopherol, phylloquinone (vitamin K1), retinal, retinoic acid, and retinol were purchase from Millipore-Sigma (St. Louis, MO, USA). Anhydrotetracycline was obtained from Cayman Chemical (Ann Arbor, MI, USA).

To determine if metabolic intermediates of the mevalonate pathway or LDL treatment revert the cells’ atorvastatin-sensitive phenotype, the statin-sensitive cancer cell line HOP-92 was seeded in 12-well plates at a density of 1 × 10⁵ cells/mL (1 mL/well), incubated overnight, and then treated with 10 μM atorvastatin and various concentrations of R-mevalonic acid (10–50 μM; Sigma-Aldrich, St. Louis, MO), ubiquinone (25–200 μM; Wako, Osaka, Japan), dolichol (75–300 μM; Avanti Polar Lipids, Alabaster, AL), squalene (10–100 μM; Wako), FPP (1–25 μM; Echelon, Salt Lake City, UT), GGPP (1–25 μM; Sigma-Aldrich), or LDL (50–200 μg/mL; Alfa Aesar, Ward Hill, MA) for 48 hours. We chose the doses of each substrate according to published references [53 (link), 54 (link)]. In select experiments, we photographed these cells with a phase-contrast microscope to capture any morphological changes. After the incubation, cells were harvested, and cell numbers were counted using a Scepter handheld automated cell counter (Millipore). Statistical analyses were performed using one-way ANOVA and Bonferroni-Dunn post-hoc tests. P values of less than 0.05 were considered statistically significant.