Luciferase assay kit

NOX4 promoter (-2000-0) luciferase reporter plasmid was constructed adopting the GV238-REPORT vector (GENE, Shanghai, China). 293 T cells were seeded in 24-well plates and transfected with 0.5 μg of GV238 plasmid, 0.2 μg of β-gal plasmid, pENTER-Egr1 plasmid (0, 0.2, 0.4, 0.6, 0.8, and 1.0μ g), and pENTER-vector plasmid (1.0, 0.8, 0.6, 0.4, 0.2, and 0 μg). Lipofectamine® 3000 (Invitrogen, CA, USA), OptiMEM (Gibco, CA, USA), and β-gal were transfected as a transfection control. Cells were harvested 48 h after transfection and analyzed adopting luciferase assay kits (Beyotime, China). All experiments were performed in triplicate.

To detect constitutive activity of BRS3, HEK293 cells were seeded in 24-well plates and cotransfected with the CRE/NFAT-RE/SRF-RE/SRE luciferase reporter plasmids (50 ng), the BRS3 from different species, or BRS3 mutants with different doses (10 ng/50 ng/150 ng); after 24 h, cells were further incubated in serum-free media for another 12 h before luciferase assay. As for peptide supplementation treatment, HEK293 cells were cotransfected with the CRE/NFAT-RE/SRF-RE/SRE luciferase reporter plasmids (50 ng) and various genes (300 ng) of the bombesin receptor family. After 24 h, cells were further incubated in serum-free media and GRP/NMB peptides (0/10/100/1,000 nM) or the N-terminal peptides of mBRS3 (1,000 nM) with different concentrations for another 12 h. Luciferase activities were determined using luciferase assay kits (Beyotime, Shanghai, China) and normalized to β-galactosidase activity. All experiments were performed at least three times in triplicates. HEK293 cells transfected with pcDNA were used as a blank control in all luciferase experiments, and the fold was calculated compared to blank control.

To elucidate the pharmacological properties of grass carp GPR84 (ciGPR84), we employed DIM as a representative agonist, known for its potent activation of GPR84 in mammalian systems. Additionally, considering their potential endogenous role, we investigated the activation potency of three commonly occurring MCFAs in gastrointestinal tracts: capric acid, undecanoic acid, and lauric acid. These chemicals were obtained commercially from Shanghai Macklin Biochemical Technology (Shanghai, China), and their purities are as follows: DIM (purity ≥ 98%), capric acid (purity ≥ 99.5%), undecanoic acid (purity ≥ 99%), and lauric acid (purity ≥ 99.5%).
Since the aforementioned compounds are insoluble in water, they were initially dissolved in 3% dimethyl sulfoxide (DMSO), then coated with 2% bovine serum albumin (BSA) to create a stock solution with a concentration of 10 mM. Prior to use, the stock solution was diluted to the working concentration using a serum-free DMEM medium. The concentration of fatty acids in the solution was detected using a Tecan F200 microplate reader (Tecan, Männedorf, Switzerland) through colorimetric analysis. The luciferase assay kits were obtained from Beyotime Biotechnology (Shanghai, China). The plasmids pGL4.29 and pGL4.33 used for examining signaling were obtained from Promega (Madison, WI, USA).