Figure 4.

Glycine Potentiates PMO Activity by Replenishing One-Carbon Unit Pool

(A) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from mdx mice treated with PMO-G and bitopertin (BP) (scale bar, 100 μm). Glycine or the mixture of glycine and BP was administered into mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (B) 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). (C) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (D) 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 and other solutions was injected into mdx TA muscles and muscles were harvested 2 weeks later. (E) 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 from muscle samples from untreated and treated mdx mice were loaded. (F) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from treated mdx mice (scale bar, 100 μm). Glycine, formate, tetrahydrofolate (THF), or the mixture of glycine with methotrexate (MTX) or THF was injected in mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (G) 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). (H) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (I) 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). (J) 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). 0.5, 5, and 10 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.