Overexpression of Clock, Bmal1, Per1, and Per2

Mouse Clock, Bmal1, Per1, and Per2 cDNAs were inserted into the pMXs-IP vector (Kitamura et al., 2003). PLAT-E cells (Morita et al., 2000) were seeded at a density of 2.5 × 10⁵ cells/well in a 12-well plate with 10% FBS on day 1. On day 2, cells were transfected with 750 ng of the pMXs-IP vectors with 2.3 µl FuGENE 6 (Promega; E2691). The culture medium was replaced with fresh DMEM with 10% FBS, and C2C12 cells were seeded at a concentration of 10⁵ cells/well in a 12-well plate on day 3. PLAT-E cell medium containing the virus was harvested on day 4 and filtered through a 0.45-µm syringe filter before transduction into C2C12 cells with 8 µg/ml polybrene (MilliporeSigma; H9268). The medium was replaced again on day 4 before the second transduction on day 5. Starting on day 6, 1 µg/ml puromycin dihydrochloride selection for virus-integrated cells was performed for 5 d. Proliferating cells were frozen in liquid nitrogen or used in experimentation.