Transactivation of MyoD Luciferase Constructs

The PGL2 luciferase reporter vectors containing the MyoD promoter, MyoD PRR, MyoD DRR, and MyoD CE are described in detail in [29 (link)]. Renilla plasmid (Renilla Luciferase Assay System, Promega, Madison, WI, USA) was used as an internal control. For transfections, 2 × 10⁶ C2C12 cells were allowed to attach overnight, medium was replaced with Opti-MEM, and the following plasmids were transfected: (0.25 µg) MyoD, MyoD PR, MyoD CE, MyoD DRR (0.04 µg) Renilla, (0.5 µg) β-m, and SKIIP, (0.25 µg) using 2 µL of lipofectamine (Invitrogen, Carlsbad, CA, USA) in 100 µL of serum-free medium. lipofectamine-DNA binding was allowed to proceed for 15 min, and then, the mixture was added to the cells. After 4 h, appropriate amounts of serum were added, and the cells were allowed to grow for 48 h, scraped and washed first with PBS and then with 250 mM Tris-HCl buffer, pH 7.2. Total cell extracts were prepared by treating the cells with lysis buffer (Pierce, Rockford, Il, USA), and transactivation of MyoD luciferase constructs was determined using the luciferase kit from Pierce as per the manufacturer’s protocol. The activity of the Renilla luciferase was used for normalizing the transfection efficiency. The results presented are the average of three experiments.