Journal: Metabolites

Article Title: The Cholesterol Metabolite Cholest-5-en-3-One Alleviates Hyperglycemia and Hyperinsulinemia in Obese ( db / db ) Mice

doi: 10.3390/metabo12010026

Figure Lengend Snippet: Luciferase activities in NFκB-Luc/CHO-K1 cells. Cells were treated with or without murine TNFα (10 ng/mL in DMSO), cholesterol (100 µM in DMSO), 5-cholestenone (100 µM choles-5-en-3-one in DMSO), or 1 µM IKK2 inhibitor IV. After 7 h of incubation, NFκB luciferase was detected using the ONE-Step TM Luciferase assay system. Values are expressed as the mean ± standard error ( n = 3 wells). * Significant difference at p < 0.05 compared to DMSO (TNFα-) treatment. † Significant difference at p < 0.05 compared to DMSO (TNFα+) treatment.

Article Snippet: In the NFκB-Luc/CHO-K1 cell line (BPS Bioscience, San Diego, CA, USA), fLUC expression is controlled by the NFκB response element located upstream of the TATA promoter and is suitable for monitoring the activity of the NFκB transcription factor through luminescence readout.

Techniques: Luciferase, Incubation

Journal:

Article Title: Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G)

doi: 10.1194/jlr.M800625-JLR200

Figure Lengend Snippet: Phenolic acids stimulate 35S-GTPγS binding to membranes from cells transfected with GPR109A. Ligand-induced 35S-GTPγS binding was studied using membranes prepared from CHO-K1 cells expressing human GPR109A (A), GPR109B (B), or GPR81 (C). Binding of 35S-GTPγS was determined in the presence of increasing concentrations of nicotinic acid (closed circles), acifran (triangles), trans-cinnamic acid (closed diamonds), p-coumaric acid (open circles), o-coumaric acid (squares), benzoic acid (open diamonds), or salicylic acid (crosses). Data are shown as the means ± SD of triplicates of a representative experiment. Three to four independent experiments were performed with similar results.

Article Snippet: Membranes prepared from Chinese Hamster Ovary (CHO)-K1 cells stably expressing GPR109A, GPR109B, GPR81, PUMA-G, or vector control (7 μg/assay) were diluted in assay buffer (100 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4) in Wallac Scintistrip plates (PerkinElmer, Shelton, CT) and preincubated with test compounds diluted in assay buffer containing 40 μM GDP (final [GDP] was 10 μM) for ∼10 min before addition of 35 S-GTPγS to 0.3 nM.

Techniques: Binding Assay, Transfection, Expressing

Journal:

Article Title: Phenolic acids suppress adipocyte lipolysis via activation of the nicotinic acid receptor GPR109A (HM74a/PUMA-G)

doi: 10.1194/jlr.M800625-JLR200

Figure Lengend Snippet: Phenolic acids compete with 3H-nicotinic acid binding to membranes from cells transfected with GPR109A. Binding of 50 nM 3H-nicotinic acid to membranes prepared from CHO-K1 cells expressing human GPR109A was studied in the presence of increasing concentrations of A: nicotinic acid (closed circles) or the cinnamic-acid-related compounds, including trans-cinnamic acid (closed squares), p-coumaric acid (open circles), o-coumaric acid (open squares), and caffeic acid (crosses), or B: nicotinic acid (closed circles) or the benzoic-acid-related compounds, including benzoic acid (closed triangles), p-hydroxybenzoic acid (open triangles), salicylic acid (open diamonds), and protocatechuic acid (closed diamonds). Data are shown as the means ± SD of triplicates of a representative experiment.

Article Snippet: Membranes prepared from Chinese Hamster Ovary (CHO)-K1 cells stably expressing GPR109A, GPR109B, GPR81, PUMA-G, or vector control (7 μg/assay) were diluted in assay buffer (100 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4) in Wallac Scintistrip plates (PerkinElmer, Shelton, CT) and preincubated with test compounds diluted in assay buffer containing 40 μM GDP (final [GDP] was 10 μM) for ∼10 min before addition of 35 S-GTPγS to 0.3 nM.

Techniques: Binding Assay, Transfection, Expressing