Dual luciferase reporter gene assay system kit

The DF-1 cell line is the most investigated and widely used chicken cell line, and its culture is the same as that reported by Himly et al.; these cells were used for the dual-luciferase reporter assay [51 (link)]. When the DF-1 cell density was ≥ 70%, the gga-miR-449b-5p mimics or NC was cotransfected with IGF2BP3 WT or IGF2BP3 MUT and MMP2 WT or MMP2 MUT for 36 h using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). Firefly and sea kidney luciferase activities were detected by the Dual Luciferase Reporter Gene Assay System Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

PK-15 cells (Procell, Wuhan, China) cultured in 24-well plates were transfected using Lipofectamine™ 3000 Transfection Reagent (Thermo, USA) according to the manufacturer's instructions. The transfection mixture for each well contained 250 ng 3′-UTR luciferase reporter vector, 12.5 ng of pGL4.74 (Promega, USA) for normalization, and either 15 pmol miR-331-3p mimic expression plasmids, miR-331-3p mimic, or negative control mimic (GenePharmn, Shanghai, China). Each group of experiments was repeated at least 3 times. After 48 h of transfection, the luciferase activity was measured using the Dual-luciferase Reporter Gene Assay System Kit (Promega).

A luciferase reporter assay was used to investigate the effect of MYH11 on the transcriptional activity of the TNFRSF14 promoter. The potential binding sites between MYH11 and the TNFRSF14 promoter were obtained from hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/). The TNFRSF14 promoter sequence containing the binding site was inserted into the pGL3-basic vector (Promega Corporation, Madison, WI, USA) to construct a luciferase reporter vector. The above vector plasmids were co-transfected with oe-NC or oe-MYH11 into 293 T cells (ATCC) using Lipofectamine 2000, respectively. After 48 h of transfection, luciferase assays were performed using the Dual Luciferase Reporter Gene Assay System kit (Promega).