Figure 6.

Glycine Augments PMO Activities by Heightening mTORC1 Activation in mdx Mice

(A) Western blot to detect phosphorylated mTOR, S6K1, and S6 expression in quadriceps from mdx mice treated intravenously with PMO-S or PMO-G at 25 mg/kg/week for 3 weeks with additional supply of glycine every other day for 5 weeks. α-Actinin was used as the loading control. 50 μg of total protein was loaded. (B) Quantitative analysis of the ratio of phosphorylated mTOR, S6K1, and S6 to mTOR, S6K1, and S6 total protein expression, respectively (n = 3; two-tailed t test). (C) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from treated mdx mice (scale bar, 100 μm). Glycine (Gly) or the mixture of glycine with PP242 (Gly/PP242) was injected into mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (D) Quantitative analysis for PAX7⁺ and PAX7⁺/Ki67⁺ MuSCs in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (E) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test) (scale bar, 100μm). (F) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). PMO (2 μg) in saline (PMO-S), glycine (PMO-G), or the mixture of glycine with PP242 (PMO-G/PP242) was injected into mdx TA muscles and muscles were harvested 2 weeks later. (G) Western blot and quantitative analysis for dystrophin expression in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). 2.5 and 5 μg of total protein from C57BL/6 mice and 50 μg of muscle samples from untreated and treated mdx mice were loaded. (H) Hierarchical clustering analysis of cell cycle-related gene expression profiles in primary myoblasts isolated from mdx mice and then treated with 0.8 mM glycine for 24 h. Expression levels (fold) are depicted in colors in which red represents upregulation and green means downregulation. (I) Quantitative real-time RT-PCR analysis of cell cycle-related gene expression in TA muscles from mdx mice treated with glycine every other day for 1 week intravenously (n = 3; two-tailed t test). CDK1, cyclin-dependent kinase 1; CDC20, cell division cycle 20; CCNB2, cyclin B2; CCNB1, cyclin B1; CCNA1, cyclin A1; CCNE1, cyclin E1. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.