Firefly renilla dual luciferase reporter assay system

The 3’UTR of wild‐type or mutant DRP1, an assumed miR‐575 binding site, was ligated into the pmirGLO luciferase vector (Promega, Madison, WI, USA). HNC cells were co‐transfected with miR‐575 mimic (or negative control) and the DRP1‐wild‐type or DRP1‐mutated by Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA). After transfection 36 h, the luciferase activities were detected using a dual‐luciferase reporter system (Promega) according to the manufacturer’s instruction. A 600‐bp DNA fragment spanning +1 to −600 of MMP12 promoter containing FOXM1 binding site was amplified and cloned into pGL3 basic vector. The promoter sequence was validated by sequencing. For examining promoter activity, cells were cotransfected with pGL3‐basic vector or pGL3‐MMP12 and internal control pRL‐TK plasmid (250 ng·mL⁻¹) with/without Flag‐FOXM1 expression vector using Lipofectamine 3000. Following cell lysis (36 h post‐transfection), Firefly and Renilla luciferase activities were evaluated in the Firefly/Renilla Dual Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). All reporter gene assays were performed in triplicate and repeated at least 3 times.

The effect of CDK2 on the transcriptional activity of E2F1 in HEK293T and A549 cells was determined by a dual-luciferase report assay using CDK1 promoter as substrate [42 (link)]. The human gene (CDK1) promoter was sub-cloned into pGL3 vector (Promega, Madison, WI, USA) in HEK293T or A549 cells. Co-transfection of gene promoter vector pcDNA3.1-E2F1 or pcDNA3.1-E2F1+pcDNA3.1-CDK2 was conducted, and a luciferase assay was performed 48 h after transfection using the Firefly/Renilla Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Cell transfections with the luciferase reporter plasmids were performed with LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 2 × 10⁵ HUVECs were co-transfected with 2 μg of HRE-driven luciferase reporter and 0.1 μg of Renilla luciferase reporter as the internal control followed by subjecting to PM2.5 exposure. Luciferase activity was tested using Firefly-Renilla Dual-Luciferase Reporter Assay System (Promega). The data were obtained by normalizing the activity of the experimental reporter to that of the internal control. The results were presented as the relative induction by normalizing the luciferase activity in the PM2.5-treated cells to the luciferase activity in untreated control cells, as previously described^{17 (link)}.