Luciferase reporter gene test kit

C2C12 cells and HEK293 cells were transfected using the polyethyleneimine (PEI, #25414; Polysciences, Warrington, UK) transient transfection method, with empty vector-transfected cells used as a control group. Cells were cultured to a density of 50–70% when the plasmids were transfected and changed to fresh medium after 4–6 h. To determine luciferase reporter gene activity, CypA and Runx2 expression constructs, luciferase reporter genes (ALP-Luc, BSP-Luc, OC-Luc, OSE-Luc) 0.3 μg, and β-galactosidase plasmid (internal control) 0.05μg were co-transfected into C2C12 cells. Twenty-four hours after transfection, the cells were treated with BMP4 and the indicated luciferase assay was performed using a luciferase reporter gene test kit (#E1501; Promega, Madison, WI, USA) to measure luciferase activity with a TriStar ² Multimode Reader apparatus. All experiments were performed in triplicate.

TargetScan databases (https://www.targetscan.org/vert_80/) predicted potential miR-21-5p binding sites to PTEN. A dual luciferase reporter assay was used to determine the potential relationship further. PTEN wild-type and mutant 3'-UTR sequences were cloned into the vector containing the luciferase reporter gene separately. Cells were collected 48 h after transfection according to the instruction for the luciferase reporter gene test kit (Promega, USA).