Renilla luciferase reporter

We seeded 293T cells in 24-well plates, cultured then overnight, and transfected them with the wildtype and mutant TRPM7 promoter-luciferase plasmids (0.1 μg per well of pGV272-wt-TRPM7 or pGV272-mt-TRPM7 plasmids) mediated by Lipofectamine 2000 (Invitrogen, CA, United States). Meanwhile, cells were cotransfected with either 0.4 μg miR-21 mimics or 0.4 μg miR-21 NC (Genepharma, Shanghai, China). Transfection efficiency was standardized by cotransfection with 0.02 μg Renilla luciferase reporter (Genechem, Shanghai, China). Both firefly and Renilla luciferase activities were quantified using a dual-luciferase assay system (Promega, E1910).

HEK 293 cells were seeded in 24-well plates at approximately 10⁵ per well and cultured overnight. The wildtype or mutant AKT promoter-luciferase plasmids (0.1 μg per well of pYr-rat-AKT-3UTR or pYr-rat-AKT-3UTR-mut plasmids) and wildtype or mutant CDK2 promoter-luciferase plasmids (0.1 μg per well of pYr-rat-CDK2-3UTR or pYr-rat-CDK2-3UTR-mut plasmids) were transfected to different groups of cells mediated by Lipofectamine 2000 (Invitrogen). Cells were also cotransfected with either 0.4 μg miR-21-3p mimics or 0.4 μg miR-21-3p NC accordingly (Genepharma). Cotransfection with 0.02μg of Renilla luciferase reporter was used to standardize the transfection efficiency (Genechem). Luciferase activities were quantified using a dual-luciferase reporter assay system (Promega). All the experiments were executed in triplicate.