Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy

Figures

• 

  Figure 1. mRNA expression of fibrosis-related genes in the DIA muscle from C57BL/10 and mdx mice. RT-qPCRs for (a) Postn, (b) Tgf-β1, (c) Ctgf and (d) Col1α1 were performed on RNA harvested from the DIA muscle of male C57BL/10 and mdx mice at 3, 5, 8 and 12 months of age. Relative levels of expression were determined by normalising to the expression level of Rplp0. Data shown as means ± S.E.M., n = 5 mice per age group and strain. No outliers were identified, and statistical analysis was performed using unpaired Student’s t test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****) between genotypes and timepoints.

• 

  Figure 2. Periostin and Tgf-β1 protein expression in C57Bl/10 and mdx mouse DIAs at 3, 5, 8 and 12 months of age. All samples for each age group for each strain were homogenised, and total protein was extracted. In total, 10 µg total protein was separated on a 4–12% Bis-Tris gel followed by a transfer onto a 0.45 µm nitrocellulose membrane. A Chameleon Duo protein ladder (Li-Cor) was used to assess the protein size. The membranes were imaged using the Odyssey CLx system and analysed using ImageStudio software, version 5.2. (a) Combined Western blot images of all samples for each age group for periostin, Tgf-β1 and vinculin housekeeping protein expression in the DIAs of C57BL/10 and mdx mice at 3, 5, 8 and 12 months of age. (b) The relative periostin and Tgf-β1 protein expression was determined via semi-quantitative analysis. The levels of protein were normalised to that of vinculin to determine the relative protein levels. Protein quantification was performed using ImageStudio Software (Li-Cor). Data shown as means ± S.E.M., n = 5 mice per age and model unless otherwise stated. Statistical analysis was performed using unpaired Student’s t test, p < 0.01 (**), p < 0.001 (***),and outliers…

• 

  Figure 3. Immunohistological analysis of fibrosis in C57BL/10 and mdx mouse DIAs. The levels of muscle fibrosis in the DIAs of 5-month-old C57BL/10 and mdx mice were assessed through immunostaining for periostin or Sirius red staining (for collagen 1 and 3). Representative images are shown for (a) periostin, with periostin stained in red, laminin in green and DAPI in blue, and (b) Sirius red, with collagen 1 and 3 stained in red. The relative quantifications of (c) periostin and (d) collagen positive areas are shown as the percentages of the total areas of the corresponding images. Data shown as means ± S.E.M., n = 4 muscles/mouse model. Statistical analysis was performed using Student’s t test, p < 0.01 (**), p < 0.001 (****).