Circadian regulation of the Igf2 gene and C2C12 cell differentiation. (A) Locations of the primers used in the 3C experiments and BamHI sites in relation to the Igf2 promoter 3 and enhancer. The primer shown in red was used in combination with one of the primers shown in black in 3C, and the results were plotted in B and C. (B and C) Relative cross-linking frequency obtained with 3C comparing different time points (B) and Per KO cells and control (C). The value obtained with the Clock gene primers was defined as 1.0. (D) Schedule of circadian synchronization and initiation of differentiation. After incubation with dexamethasone between −1 h and 0 h, the culture medium was replaced with growth medium containing 10% FBS at 0 h. The culture medium was replaced with differentiation medium (DM) containing 5% HS at different time points every 4 h (arrows). Differentiation was continued for 48 h from each starting point before fixation or harvest for various analyses. (E) Immunofluorescence staining of C2C12 cells with MHC antibody and Hoechst 48 h after starting differentiation at the indicated time points shown in D. Bar, 200 µm. (F–H) Analyses of differentiation index (F), fusion index (G), and relative expression of differentiation-specific genes (H) with C2C12 cells that were induced to differentiate at the indicated post-synchronization time points. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test comparing two time points in B, control and Per KO cells in C, and different time points in control cells in F–H. Graphs show mean ± SEM of biological triplicates. Each replicate includes n = 1,000–1,500 nuclei in F and G. ^#, 24-h rhythmicity with Cosinor (P < 0.05); ^##, P < 0.01; and ^###, P < 0.001.