Figure 1

Dystrophin restoration in heart following Pip6f-PMO treatment in mdx mice.

(A) Representative image of dystrophin immunohistochemical staining in heart following single 10 mg/kg Pip6f-PMO IV administration in mdx mice. (B) Dystrophin western blot of treated mdx hearts relative to 5% C57BL/10 following a single 10 mg/kg Pip6f-PMO administration. Approximately 5% dystrophin restoration observed. All samples run under the same experimental conditions and on the same SDS gel. Dotted line indicates where image was cropped. (C) Mice then underwent 4 daily injections followed by additional administrations every two weeks. Dystrophin immunohistochemical staining of treated heart with inserts indicating higher magnification of designated areas namely the right ventricle (RV) wall (C, i), outer left ventricle (LV) wall at apex (C, ii), inner myocardium (C, iii) and base (C iv and v) of heart. (D) Representative images of immunohistochemical staining of dystrophin protein in exercised C57BL/10, mdx and Pip6f-treated mdx mouse hearts. (E) Quantification of immunohistochemical staining in hearts (from D) following multiple administrations. Dystrophin expression is determined relative to laminin co-stain. The scatter plots show the normalised relative intensity values for each region of interest. Statistical significance was determined using ANOVA followed by Games-Howell post-hoc test to correct for variance heterogeneity (*** = P < 0.001, ** = P < 0.01, * = P < 0.05). (F) Representative images of reverse-transcriptase (RT) PCR illustrating Δ23 splicing in heart Pip6f-PMO treated cohort. All samples run under the same experimental conditions and on the same agarose gel. RT-qPCR was also performed (see Supplementary Fig. 3 B) which resulted in 32.3% Dmd transcripts lacking exon 23, following normalisation to exon 20–21 (SEM 3.1). (G) Representative images of western blots for Pip6f-PMO treated heart. For western blots, 10 μg of protein was loaded and dystrophin (dys) was quantified relative to vinculin loading control. All samples run under the same experimental conditions and on the same SDS gel. Dotted line indicates where image was cropped. For full agarose and SDS gels see Supplementary Fig. 1, 5&6.