Fig. 4

ROS regulates axonal local translation in MNs. a SUnSET assay. Representative images of WT and SMA MNs. Neurites were selected with a segmented line, straightened, and divided into 20 µm bins using the concentric circles plugin. Quantification of mean puromycin intensity profiles confirms that protein synthesis is reduced in the soma and axonal compartment of SMA MNs. b SUnSET assay. Representative images of SMA MNs after ROS modification. Tau staining shows whole neuronal morphology and puromycin signal represents newly synthesized proteins after 1 h treatment. 10 µM NAC, 100 µM menadione or 50 µM anisomycin were treated. Each dot represents the average intensity of 15 neurons (N = 5). One-way ANOVA with Tukey HSD post hoc analysis was used to determine statistical significance for multiple comparisons. Bar graphs depict the mean ± S.D. n.s. p > 0.05, ***p < 0.001. c SUnSET assay. Representative images of SMA axons. MNs were treated with 10 µM NAC, 50 mM pyruvate, 100 µM menadione, or 50 µM anisomycin for 1 h. a–c Intensity of incorporated puromycin signal is represented in rainbow scale. Scale bar: 20 µm. a, c Each dot represents the average quantification of 10 neurons of 5 individual biological replicates (N = 5). Bar graphs depict the mean ± S.D. Two-tailed unpaired t-test with Holm-Bonferroni correction for multiple comparisons was used to determine statistical significance. n.s. p > 0.05, *p < 0.05, **p < 0.01