Fig. 3

Analyses of necrotic and regenerating myofibers in the masseter and tongue muscle of WT and DMD rats (A–F). A, B Representative immunohistochemistry of endogenous IgG (red) and laminin (green) in the masseter (A) and tongue (B) muscle of DMD rats at 6 months old. Nuclei were stained with Hoechst 33258 (blue). Arrowheads (white) indicate necrotic myofibers. C, D Representative immunohistochemistry of embryonic myosin heavy chain (eMHC) (red) and laminin (green) in the masseter (A) and tongue (B) muscle of DMD rats at 6 months old. Nuclei were stained with Hoechst 33258 (blue). Arrowheads (white) indicate regenerating myofibers. E Quantitative analysis of necrotic and regenerating myofibers in masseter and tongue muscle of WT (n=5) and DMD (n=6) rats at 6 months old. The data are expressed as mean±SE. *, p<0.05 vs others by Tukey–Kramer test. Analyses of tongue muscle of WT and DMD rats (G–J). G, H Distribution of myofiber diameters at 6 months old. The left panel (G) depicts the relative distribution of myofibers with certain diameters. The right panel (H) depicts relative cumulative plots based on the data shown in the left panel. The myofiber diameters of DMD rat (n=6) were larger than those of WT rats (n=5). p<0.01 by Wilcoxon rank sum test (number of myofibers analyzed, WT=3112 and DMD=2662). I Ratio of myofibers with central nuclei at 6 months old. The data are expressed as mean±SE. **, p<0.01 by unpaired Student’s t-test. J Relative fibrotic area at 6 months old. The data are expressed as mean±SE. **, p<0.01 by unpaired Student’s t-test. Quantitative analyses of molecules involved downstream of Akt-mTOR pathway in the tongue muscle of WT (n=5) and DMD rats (n=6) at 6 months old (K–R). K–N Ratio of phosphorylated Akt, S6K (S6 kinase), S6 (S6 ribosomal protein), and 4E-BP1. O, P Total amount of Akt, S6K (S6 kinase), S6 (S6 ribosomal protein), and 4E-BP1 normalized to that of α-tubulin. The data are expressed as mean±SE. Quantitative analyses of expression of two muscle-specific E3 ubiquitin ligases (Atrogin1 and MuRF1) in the tongue muscle of WT (n=5) and DMD rats (n=6) at 6 months old (S). The data are expressed as mean±SE. Quantitative analysis in the number of nuclei per myofiber of the tongue muscle of WT (n=5) and DMD rats (n=6) at 6 months old (T). The data are expressed as mean±SE