Cell experiment:

Stock solutions of 27-Hydroxycholesterol are prepared in 100% ethanol and stored at –80℃. 27-Hydroxycholesterol stock solution is dissolved in appropriate volumes of media to prepare the working solutions of 1 μM.

Animal experiment:

Proliferation assays are conducted on black 96 well plates using a commercial kit which quantifies cell number using DNA content and membrane integrity. LNCaP and PC3 cells are treated with 1 μM 27-Hydroxycholesterol for 48 hours[2].

25(R)-27-hydroxy Cholesterol ((25R)-26-hydroxy cholesterol) is the naturally occurring isomer of the endogenous the endogenous 27-hydroxycholesterol. And approximately 80% is the naturally occurring (25R) isomer [1][2]. 27-hydroxycholesterol is an endogenous ligand of liver X receptor with EC50 values of 85 and 71 nM for LXRα and LXRβ, respectively [2][3].

The liver x receptors (LXRα and LXRβ) are nuclear hormone receptors that are important regulators of cholesterol and fatty acid metabolism [2].

27-hydroxycholesterol is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues [1]. 27-hydroxycholestero1 can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity [1]. In cholesterol-loaded human macrophage cells, 27-hydroxycholestero1 is an endogenous ligand for LXRs with Kd values of 85 and 71 nM for LXRα and LXRβ, respectively [3]. In human fibroblasts, low density lipoprotein cholesterol is converted to 27-hydroxycholesterol, which was sufficient to reduce the activity of HMG-CoA reductase [4].

References:
[1]. Javitt NB. 26-Hydroxycholesterol: synthesis, metabolism, and biologic activities. J Lipid Res. 1990 Sep;31(9):1527-33.
[2]. Fu X, Menke JG, Chen Y, et al. 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J Biol Chem. 2001 Oct 19;276(42):38378-87.
[3]. Zhang Y, Mangelsdorf DJ. LuXuRies of lipid homeostasis: the unity of nuclear hormone receptors, transcription regulation, and cholesterol sensing. Mol Interv. 2002 Apr;2(2):78-87.
[4]. Axelson M, Larsson O. Low density lipoprotein (LDL) cholesterol is converted to 27-hydroxycholesterol in human fibroblasts. Evidence that 27-hydroxycholesterol can be an important intracellular mediator between LDL and the suppression of cholesterol production. J Biol Chem. 1995 Jun 23;270(25):15102-10.