Dual luciferase reporter gene assay kit

We constructed dual-luciferase reporter plasmids containing wild-type S100A8 promoter (S100A8-WT), mutant S100A8 promoter (S100A8-Mut, sites 663–669 were changed from AGGAAGT to CTCTCTC and sites 411-416 were changed from GGGAAG to CTCTCT, Supplementary Table 1), S100A9-WT, and S100A9-Mut (sites 769–774 were changed from GGGAAG to CTCTCT, Supplementary Table 1). Thereafter, the reporter plasmids were co-transfected into primary cardiomyocytes with si-NC and si-SPI1. The cells were lysed subsequent to 48 h of transfection and 1 min of centrifugation at 13,000 g. Next, the supernatant was attained for the determination of the luciferase activity with the dual-luciferase reporter gene assay kit (16185, Thermo Scientific, Fremont, CA). With Renilla luciferase activity as an internal reference, the relative activity of luciferase was calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity.

HepG2 were seeded into 24-well plates at a density of 1×10⁵ cells/well. The cells were grown to 70% confluence in 24-well plates before transfection at 37°C. Next, cells were transfected with the pmirGLO reporter plasmid (empty plasmid) (Promega Corporation), pmirGLO reporter plasmid containing 3′-UTR of SFRP5 mRNA, miR-21-5p mimic (50 nm; 5′-GUCUCAAUAUUAGGCUAAGCUAU-3′), miR-21-5p inhibitor (50 nm; 5′-AUAGCUUAGCCUAAUAUUGAGAC-3′), mimic negative control (NC; 5′-UUCUCCGAACGUGUCACGUUU-3′) or inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′) (Shanghai GenePharma Co., Ltd), using Lipofectamine^® 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at room temperature. Subsequently, the transfected cells were cultured for 24 h at 37°C in a 5% CO₂ humid incubator. Then, the luciferase activity was detected according to the instructions of the Dual-Luciferase reporter gene assay kit (Thermo Fisher Scientific, Inc.). Renilla luciferase activity was normalized to firefly luciferase activity and the result was shown as relative luciferase activity. Transfection experiments were performed 3 times in triplicate. Data were represented as the ratios of luciferase activities/Renilla activities.