Figure 3: Genetic deletion of p47^phox decreases oxidative stress and induces activation of autophagy in mdx mice.

(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn²⁺. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47^−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition. (c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies. (d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47^−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments. (e) LC3-LAMP1 colocalization in single FDBs from mdx and p47^−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas). (f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47^−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus. (g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47^−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey’s post hoc test. *P<0.05 and **P<0.01.