Cholesterol liquicolor test

Reagents were obtained from the following sources: Cholesterol LiquiColor Test (enzymatic) from Stanbio (Boerne, TX); bicinchoninic acid assay protein kit from Pierce Biotechnology, Inc (Rockford, IL); organic solvents were from J. T. Baker (Phillipsburg, NJ); TRIzol reagent and SuperScript II were from Invitrogen (Carlsbad, CA); RNeasy kit from QIAGEN (Valencia, CA); SYBR Green TaqMan PCR kit from Applied Biosystems (Foster City, CA); corticosterone ELISA kit from Cayman Chemical (Ann Arbor, MI); progesterone and testosterone ELISA kits from ALPCO (Salem, NH); pregnant mare's serum gonadotropin (PMSG)/equine chorionic gonadotropin (eCG), human chorionic gonadotropin (hCG), and Bt₂cAMP from MilliporeSigma (St. Louis, MO). Cortrosyn was from Amphastar Pharmaceuticals, Inc (Rancho Cucamonga, CA).

HepG2 cells (5 × 10⁵ cells/well) were equally seeded in 6-well plates. The adherent cells were exposed to R and Q for 24 h. Then, the cells were extracted using a mixture of chloroform and methanol (3:1, v/v) for 30 min, the samples were then centrifuged at 13,000× g × 15 min for purification. The pellet was lysed in a RIPA buffer for measurement of protein abundance, and the supernatant was collected for total cholesterol measurement by a cholesterol liquicolor test (Stanbio, Boerne, TX, USA) using a microplate reader.