Differential regeneration efficiency of TA muscle depending on the circadian timing of the injury. (A) Size distribution of H&E-stained myofibers containing centrally located nuclei on day 4.5. TA muscle was injured with barium chloride at ZT2 or ZT14 on day 0 and harvested on day 4.5. The minimal Feret’s diameter of each myofiber was calculated. n = 8 mice with 4 males and 4 females in each group in A and B. (B) Average of the minimal Feret’s diameters of myofibers with centrally located nuclei on day 4.5. 2 and 14 at the end of each genotype indicate the injury time at ZT2 and ZT14, respectively. (C) Immunofluorescence staining of WT TA muscle injured at ZT2 and ZT14 with antibodies against eMHC and laminin on day 4.5. DNA was counterstained with DAPI. Bar, 100 µm. (D) Average of the minimal Feret’s diameters of the eMHC⁺ areas on day 4.5. n = 4 mice. (E) Immunofluorescence staining of WT TA muscle sections with the MyoD antibody and an EdU kit. TA muscle was injured with barium chloride at ZT2 or ZT14 on day 0, and EdU was injected i.p. 96 or 120 h later. The muscle was harvested 12 h later at day 4.5 or 5.5. Bar, 25 µm. (F) Frequency of positive cells for EdU uptake and MyoD staining in TA muscle sections shown in E. n = 4 mice. (G) Sirius red staining of WT TA muscle on day 14 after injury. Bar, 200 µm. (H) The area percentage of fibrosis indicated by positive Sirius red staining on days 7 and 14. Data are presented as mean + SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test. The values at ZT14 in Fig. 1 were reused in these figures.