Fig. 1

Capn3-deficient and Capn3-sufficient immortalized primary muscle cells. Genotyping: agarose gel showing the WT (Capn3^+/+) Capn3 gene band at 500 bp and the Capn3-deficient (Capn3^−/−) gene band at 300 bp. The large T gene is shown at 700 bp (a). Differentiation test performed on Capn3^+/+ and Capn3^−/− primary immortalized cell cultures. Hoechst 33342 was used to stain the nuclei and show multinucleated myotubes formation after 2 days in a low-serum medium (b). Western blots and quantification of muscle-specific proteins in myoblasts and myotubes. Myogenin and dysferlin were used to verify the myogenicity of cells. The expression of myogenin and dysferlin was normalized to vinculin loading control. Since myogenin and dysferlin expression varied between calpain-deficient and calpain-sufficient myoblasts, we have expressed increased expression of these differentiation markers in myotubes as a percentage of expression seen in myoblasts (c). Analyses were performed on n = 4 different cell culture flasks for proliferation and n = 3 for differentiation. **Significantly different with p < 0.01; *significantly different with p < 0.05