Figure 3.

Autophagy is not affected by S-nitrosylation. (a) Western blot analyses of S-nitrosothiols in total homogenates of skeletal muscles obtained from GSNOR-KO (KO) and wild-type (WT) mice, subjected to biotin-switch assay and revealed by HRP-conjugated streptavidin. Lactate dehydrogenase (LDH) was selected as loading control. Results show that even in the absence of any treatment with NO-delivering drugs, GSNOR ablation induces a significant increase of S-nitrosylated proteins. (b) Representative fluorescence microscopy images of satellite cell-derived myotubes isolated from KO and WT mice expressing LC3-conjugated green fluorescent protein (GFP-LC3) in heterozygosis. Cells were then subjected to two different autophagic stimuli: (1) they were treated for 6 h with 5 μM of the proteasome inhibitor MG132 or, alternatively, (2) allowed to grow for 6 h in a nutrient-deprived cell medium (stv). Images are representative of three independent experiments that gave similar results. Both genotypes displayed a significant and similar increase of fluorescent dots, plausibly representing autophagosomes, thereby indicating that autophagy is not impaired by S-nitrosylation