Figure 1: Inhibition of Nox2 activity reduces oxidative stress and Src kinase-mediated impaired autophagy.
(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. (c) Analysis of Rac1 and (d) Src. (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody. (f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. (g) Extracellular ROS production was assessed using Amplex-red dye. (h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+. (i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs. (k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey’s post hoc test. *P<0.05 and **P<0.01.
