Plasmid Construction and Dual-Luciferase Reporter Assay

The large yellow croaker pparγ was cloned into the pCS2 + vector (Invitrogen) ( Table 1 ). The IL1β promoter plasmid (pGL6-IL1β) and IL6 promoter plasmid (pGL6-IL6) were stored in our laboratory. The sequences of IL1β promoter and IL6 promoter were shown in Supplementary Figures 1 and 2 , respectively.

In dual-luciferase reporter assay, the activity of firefly luciferase reporter gene (pGL6-IL1β and pGL6-IL6) was detected by using luciferin as substrate, and then the activity of renilla luciferasel reporter gene was detected by using coelenterazine as substrate when the firefly luciferase was quenched. The ratio of the two luciferases was the reporter promoter activity. Before the luciferase assay, HEK293T cells were seeded into 24-well plates. We used EZ Trans Plus cell transfection reagent (LIFE iLAB BIO, China) to transfect the reporter plasmids (pGL6-IL1β, pGL6-IL6), transcription factor plasmids (pCS2-PPARγ), and pRT-TK renilla luciferase plasmids into HEK293T cells. The activity of the firefly luciferase reporter gene and the renilla luciferase reporter gene were measured on the microplate reader using TransDetect^® Double-Luciferase Reporter Assay Kit (Transgene Biotech, China). The ratio of the two readings was the fluorescence value after correcting the number of cells. The experiment was repeated four times.