(A) UCSC genome browser visualization (mm10 genome) of MYOD1 binding tags within the Bmal1 locus. (B) Representative BMAL1 western blots from C2C12 myoblasts transiently transfected with 150 ng pGEM empty vector (-) or MYOD1 expression vector (+). Densitometric values are expressed as average fold-change of BMAL1 over the Tubulin loading control which was unchanged with MYOD1 transfection ± SEM (n = 3). Results were analyzed with one-way ANOVA, * indicates a p-value less than 0.05 (p = 0.025). Bmal1P-Luc luminescence in C2C12 myotubes (2C, n = 4 biological replicates) and skeletal muscle primary myotubes (D, n = 3 biological replicates) with transient transfection of pGEM control, MYOD1, or MYOD1mut expression vectors. Luciferase activity for each co-transfection is plotted as average fold-change in relation to the pGEM empty vector control ± SEM. Results were analyzed using one-way ANOVA, ** indicates a p-value less than 0.001. # indicates a p-value less than 0.01 comparing MYOD1 vs MYOD1mut. (E) Bmal1P-Luc luminescence in electroporated skeletal muscle. Bmal1P-Luc activity was normalized to Renilla luciferase as an electroporation control with the right-leg receiving MYOD1 expression vector (red circles) and the left leg receiving the pGEM empty vector control (black circles). The p-value statistic was calculated by performing a Mann-Whitney non-parametric t-test. Note, one outlier was removed from each group based upon the Robust regression and outlier removal (ROUT) test, with a false discovery rate of <0.01. Bmal1P-Luc Dual-Luciferase activities in skeletal muscle primary myotubes (F-H) Representative Bmal1P-Luc driven bioluminescence recordings in synchronized C2C12 myotubes co-transfected with pGEM control (F, n = 7), MYOD1 (G, n = 6), or MYOD1mut (H, n = 4) expression vectors. Luminescence recordings are expressed as average counts/sec (base-line subtracted) (I) Average Bmal1P-Luc amplitudes ± SEM calculated by JTK_CYCLE from 1.5 to 4.5 days post-synchronization. Results were analyzed with one-way ANOVA, ** indicates p-value less than 0.001, # indicates p-value less than 0.002 (n = 4 biological replicates per group).