Fig. 1

LC3-II protein level in human and murine samples from SMA muscle biopsies, and human lymphoblast and fibroblast cell lines. A Muscle biopsies from Control, Pompe and SMA type I and SMA type II patients were disaggregated and protein extracts were submitted to western blot analysis using anti-LC3 antibody. Membranes were reprobed with anti-CypA antibody, used as a loading control. Graph values represent the expression of LC3-II vs CypA. B Total cell lysates of gastrocnemius from genotyped WT and mutSMA P2 (left) and P5 (right) mice were submitted to western blot analysis using anti-LC3 and anti-SMN antibodies. Membranes were reprobed using an antibody against α-tubulin. Graph values represent the expression of LC3-II vs α-tubulin at P2 and P5, and correspond to the quantification of three independent experiments ± SEM. Asterisks indicate significant differences using Student t test (**p < 0.01). B Representative immunofluorescence images of gastrocnemius sections of P2 WT and P2 mutSMA mice using an anti-LC3 (green) and an anti-Laminin (red) antibodies. Hoechst dye (blue) was used to identify nuclei. Scale bar, 20 μm. Graphs represent the mean of LC3 positive puncta measured in WT and mutSMA myofibers, corresponding to the quantification of tree independent experiments ± SEM. Asterisks indicate significant differences using Student t Test (****p < 0.0001). C Protein extracts from fibroblast and lymphoblast cell lines were submitted to western blot analysis using anti-LC3 and anti-SMN antibodies. Membranes were reprobed with anti-α-tubulin antibody, used as a loading control. Graph values represent the expression of LC3-II versus α-tubulin. C Control (unaffected) and SMA II and SMA I patient fibroblast cell lines were plated and maintained in supplemented MEM. Forty-eight hours after plating, cell lysates were obtained and submitted to western blot using anti-LC3 and anti-SMN antibodies. Membranes were reprobed with an anti-α-tubulin antibody. Graph values represent the expression of LC3-II versus α-tubulin and correspond to the quantification of six independent experiments. Asterisks indicate significant differences using one-way ANOVA with Tukey’s multiple comparisons post-test (***p < 0.001, **p < 0.001). Representative immunofluorescence images of 2-day cultured Control, SMA II, and SMA I fibroblasts using anti-LC3 (green), anti-SMN (red) antibodies and Hoechst staining (blue). Hoechst was used to identify fibroblast nuclei. Scale bar, 25 µm. Graph represents the mean of LC3 positive puncta per cell and corresponds to the quantification of three independent experiments ± SEM. Asterisks indicate significant differences using one-way Anova with Tuckey’s multiple comparisons post-test (*p < 0.05; ****p < 0.0001)