Figure 2.

Effects of Early Exon-Skipping Treatment on Dystrophin Production in CXMD_J Skeletal Muscles

(A) Representative RT-PCR image showing exon 6–9-skipped bands across skeletal muscles as a result of treatment with the 4-PMO cocktail. For non-treated (NT) and treated samples, native mutant (exons 5-6-8-9-10) and skipped (exons 5–10) bands are indicated; the rest are intermediate skipping products. Specifically for NT, the band below the native band is due to spontaneous exon 9 skipping. For wild-type (WT), the topmost band is the native (exons 5-6-7-8-9-10) band, while the lower band has exon 9 spontaneously skipped. Calculated exon-skipping efficiencies are shown below. Error bars, SEM; *p ≤ 0.05 versus NT, one-way ANOVA, Dunnett’s multiple comparisons test. Dots represent individual dogs. (B) Representative western blot showing dystrophin rescue in treated CXMD_J skeletal muscles (12303MA). For non-treated and treated muscle samples, 40 μg of protein was loaded. Dystrophin was detected using DYS1; desmin and myosin heavy chain (MyHC) were detected as loading controls. Dystrophin rescue was quantified relative to 5% WT levels and is shown below. The band observed below the rescued dystrophin band is likely due to the high amount of total protein loaded. Dots represent individual dogs. Error bars, SEM. (C) Representative immunohistochemistry images indicate that the restored dystrophin (green, DYS1) in PMO-treated skeletal muscles localizes correctly to the sarcolemma; blue, nuclei. Total magnification, 200×; scale bar, 100 μm. For all panels, n = 5–6 (PMO-treated dogs). Abbreviations: TA, tibialis anterior; GRA, gracilis major; GAS, gastrocnemius; BF, biceps femoris; QUA, quadriceps; EDL, extensor digitorum longus; SOL, soleus; ECU, extensor carpi ulnaris; BIC, biceps brachii; IC, intercostal muscles; DIA, diaphragm; SC, sternocleidomastoid; ESOP, esophagus.