Figure S5.
Per1/Per2–Igf2 axis and circadian timing–dependent myogenesis and muscle regeneration.(A) ChIP-PCR analyses of indicated proteins in control and Per KO C2C12 cells at regions R1 and R3 shown in Fig. 5 F. Relative abundance compared with control IgG is shown. Peak time points when the control cell values were higher than those of Per1 KO and Per2 KO cells are highlighted with asterisks. (B) Locations of the PCR primers specific to two variants and common to all three variants of Igf2. (C) qPCR results of the Igf2 variant mRNAs in control (Cont) and Per KO cells. The PCR products obtained with the common primers largely represented the expression levels of variant 3 because the levels of variants 1 and 2 were by far lower than the level of variant 3. (D) ChIP-PCR analyses of the indicated proteins in control and Per KO C2C12 cells at regions R4 and R6 in Fig. 5 H. Relative abundance compared with control IgG is shown. Peak time points when the control cell values were higher than those of Per1 KO and Per2 KO cells are highlighted with asterisks. Pol II, RNA polymerase II. (E–G) Analyses of differentiation index (E), fusion index (F), and relative expression levels of differentiation-specific genes (G) with C2C12 cells that were induced to differentiate at the indicated post-synchronization time points. Control and Igf2 KD cells prepared with two shRNA clones were compared. (H) Average Feret’s diameters of myofibers with centrally located nuclei on day 14. TA muscle was injured with barium chloride at ZT2 or ZT14 on day 0 and harvested 14 d later. Mean + SEM of 8 mice, including 4 males and 4 females in each group, is shown. 2 and 14 at the end of each genotype indicate the injury time at ZT2 and ZT14, respectively. Data are presented as mean + or ± SEM of biological triplicates. Each replicate includes n = 1,000–1,500 nuclei in E and F. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test compared with control values in A and D, control common primers in B, and different time points in control cells in E–G. #, 24 h rhythmicity with Cosinor (P < 0.05); ##, P < 0.01; and ###, P < 0.001.

Per1/Per2–Igf2 axis and circadian timing–dependent myogenesis and muscle regeneration. (A) ChIP-PCR analyses of indicated proteins in control and Per KO C2C12 cells at regions R1 and R3 shown in Fig. 5 F. Relative abundance compared with control IgG is shown. Peak time points when the control cell values were higher than those of Per1 KO and Per2 KO cells are highlighted with asterisks. (B) Locations of the PCR primers specific to two variants and common to all three variants of Igf2. (C) qPCR results of the Igf2 variant mRNAs in control (Cont) and Per KO cells. The PCR products obtained with the common primers largely represented the expression levels of variant 3 because the levels of variants 1 and 2 were by far lower than the level of variant 3. (D) ChIP-PCR analyses of the indicated proteins in control and Per KO C2C12 cells at regions R4 and R6 in Fig. 5 H. Relative abundance compared with control IgG is shown. Peak time points when the control cell values were higher than those of Per1 KO and Per2 KO cells are highlighted with asterisks. Pol II, RNA polymerase II. (E–G) Analyses of differentiation index (E), fusion index (F), and relative expression levels of differentiation-specific genes (G) with C2C12 cells that were induced to differentiate at the indicated post-synchronization time points. Control and Igf2 KD cells prepared with two shRNA clones were compared. (H) Average Feret’s diameters of myofibers with centrally located nuclei on day 14. TA muscle was injured with barium chloride at ZT2 or ZT14 on day 0 and harvested 14 d later. Mean + SEM of 8 mice, including 4 males and 4 females in each group, is shown. 2 and 14 at the end of each genotype indicate the injury time at ZT2 and ZT14, respectively. Data are presented as mean + or ± SEM of biological triplicates. Each replicate includes n = 1,000–1,500 nuclei in E and F. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test compared with control values in A and D, control common primers in B, and different time points in control cells in E–G. #, 24 h rhythmicity with Cosinor (P < 0.05); ##, P < 0.01; and ###, P < 0.001.

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