luciferase reporter gene assay hek 293 cells  (BPS Bioscience)

Journal: Frontiers in Pharmacology

Article Title: Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

doi: 10.3389/fphar.2017.00468

Figure Lengend Snippet: Concentration–response studies with plant extracts regarding TGR5 activity. HEK-293 cells were cotransfected with a TGR5 expression plasmid, a CRE luciferase reporter plasmid and an EGFP plasmid as internal control. Cells were treated with 10 μM lithocholic acid (LCA) as positive control or the indicated concentrations of (A) PdioE, (B) SaroE, or (C) KgalE for 18 h. The measured luciferase-derived luminescence was normalized to the obtained EGFP-derived fluorescence. Results are expressed as % activation compared to the reference LCA. Each bar represents the mean ± SD of at least three independent experiments performed in quadruplicate and evaluated by one-way ANOVA with the Bonferroni post-test. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05 compared with solvent vehicle control (DMSO), ns not significant versus DMSO.

Article Snippet: For the TGR5 luciferase reporter gene assay HEK-293 cells (ATCC, Manassas, VA, United States) were maintained in DMEM containing 4.5 g/L glucose supplemented with 2 mM glutamine, 10% FBS, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin.

Techniques: Concentration Assay, Activity Assay, Expressing, Plasmid Preparation, Luciferase, Control, Positive Control, Derivative Assay, Fluorescence, Activation Assay, Solvent

Journal: Frontiers in Pharmacology

Article Title: Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

doi: 10.3389/fphar.2017.00468

Figure Lengend Snippet: Microbial transformation of chenodeoxycholic acid (CDCA) to lithocholic acid (LCA) increases TGR5 activity. CDCA was incubated with (CDCA FS ) or without fecal microbiota (CDCA 0 ) from mice for 24 h. Corresponding vehicle controls are labeled “- FS ” and “- 0 ”, respectively. After centrifugation and sterile filtration, a TGR5 luciferase reporter gene assay was performed as described in the Section “Materials and Methods.” Cells were treated with 50 μM of CDCA 0 and CDCA FS , respectively, for 18 h. The measured luciferase-derived luminescence was normalized to the obtained EGFP-derived fluorescence. Results are expressed as fold activation compared to the vehicle control. Each bar represents one experiment performed in octuplicates.

Article Snippet: For the TGR5 luciferase reporter gene assay HEK-293 cells (ATCC, Manassas, VA, United States) were maintained in DMEM containing 4.5 g/L glucose supplemented with 2 mM glutamine, 10% FBS, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin.

Techniques: Transformation Assay, Activity Assay, Incubation, Labeling, Centrifugation, Sterility, Filtration, Luciferase, Reporter Gene Assay, Derivative Assay, Fluorescence, Activation Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

doi: 10.3389/fphar.2017.00468

Figure Lengend Snippet: Fractionation of KgalE could not locate TGR5 activity in a specific fraction. HEK-293 cells were cotransfected with a TGR5 expression plasmid, a CRE luciferase reporter plasmid and an EGFP plasmid as internal control. Cells were treated with 10 μM lithocholic acid (LCA) as positive control or the 30 μg/mL of KgalE fractions (F1–F6) or essential oil (AEO) for 18 h. The measured luciferase-derived luminescence was normalized to the obtained EGFP-derived fluorescence. Results are expressed as % activation compared to the reference LCA. Each bar represents the mean ± SD of three independent experiments performed in quadruplicate and evaluated by unpaired Student’s t -test. ∗∗ p < 0.01, ∗ p < 0.05 compared with control (DMSO); ns, not significant versus control.

Article Snippet: For the TGR5 luciferase reporter gene assay HEK-293 cells (ATCC, Manassas, VA, United States) were maintained in DMEM containing 4.5 g/L glucose supplemented with 2 mM glutamine, 10% FBS, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin.

Techniques: Fractionation, Activity Assay, Expressing, Plasmid Preparation, Luciferase, Control, Positive Control, Derivative Assay, Fluorescence, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

doi: 10.3389/fphar.2017.00468

Figure Lengend Snippet: Pentacyclic triterpene acids in the extracts of S. aromaticum (SaroE) and P. dioica (PdioE) are the components activating TGR5. HEK-293 cells were cotransfected with a TGR5 expression plasmid, a CRE luciferase reporter plasmid and an EGFP plasmid as internal control. Cells were treated with 10 μM lithocholic acid (LCA) as positive control or 100 μg/mL of extracts (Extr) or reconstituted TTA mixtures (Mix) for 18 h. The measured luciferase-derived luminescence was normalized to the obtained EGFP-derived fluorescence. Results are expressed as % activation compared to the reference LCA. Each bar represents the mean ± SD of four independent experiments performed in quadruplicate and evaluated by unpaired Student’s t -test. ∗∗∗ p < 0.001, ∗∗ p < 0.01 compared with solvent vehicle control (DMSO); ns, not significant versus indicated group.

Article Snippet: For the TGR5 luciferase reporter gene assay HEK-293 cells (ATCC, Manassas, VA, United States) were maintained in DMEM containing 4.5 g/L glucose supplemented with 2 mM glutamine, 10% FBS, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin.

Techniques: Expressing, Plasmid Preparation, Luciferase, Control, Positive Control, Derivative Assay, Fluorescence, Activation Assay, Solvent

Journal: Molecular pharmacology

Article Title: Computer-Aided Discovery, Validation, and Mechanistic Characterization of Novel Neolignan Activators of Peroxisome Proliferator-Activated Receptor γ

doi: 10.1124/mol.109.062141

Figure Lengend Snippet: TABLE 1

Article Snippet: PPAR Luciferase Reporter Gene Transactivation HEK-293 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM with phenol red supplemented with 584 mg/ml glutamine, 100 U/ml benzylpenicillin, 100 μ g/ml streptomycin, and 10% fetal bovine serum.

Techniques: Luciferase, Expressing, Plasmid Preparation