Fig. 1

a SOD1 immunofluorescence in HEK293 cells overexpressing mutant SOD1; percentage of cells with SOD1 foci. b HRI, PKR, PERK or GCN2 knock-down effect on the percentage of HEK293 cells with SOD1 foci. HEK293 cells co-transfected with plasmids expressing SOD1 (WT or G93A) and the Cas9 endonuclease with gRNAs targeting HRI, PKR, PERK or GCN2 or a non-targeting gRNA (control). c, d PERK and GCN2 pharmacological inhibition (PERKib, GCN2ib) effect on SOD1 (WT, G93A) foci formation in HEK293 cells. FociCount SOD1 foci analysis (Fig. S2), 3 independent experiments, ≥ 100 cells/condition/experiment, one-way ANOVA and Sidak´s post-hoc test. e SOD1 and MAP2 immunofluorescence in primary neurons expressing mutant SOD1 +/-ISRIB or GCN2ib ; clustered distribution pattern quantification. One-way ANOVA and Sidak´s post hoc test, 3 independent experiments, > 25 neurons/condition/experiment. f Longitudinal tracking of neurons expressing mCherry (Ch) and Ch-tagged mutant SOD1 (G93ACh). Green arrows point to neurons tracked till the end of the experiment. Red arrows indicate neurons that died before. g Cumulative death hazard of neurons co-expressing G93ACh or Ch and gRNAs-containing CRISPR/Cas9 plasmids for GCN2 targeting (GCN2g1, GCN2g2) or px458 empty vector (ctrl). h Cumulative death hazard of neurons expressing G93ACh +/- GCN2 pharmacological inhibitor (A92). Cox Proportional Hazard analysis; pooled data from 3–5 experiments (Tables S1-S2). n; number of neurons. i EMG activations in TA from WT and G93A mice +/- GCN2ib (10 and 12 weeks of age). j Longitudinal EMG analysis in TA and SOL from WT and G93A mice +/- GCN2ib. Red/green boxes highlight differences in GCN2ib-treated G93A mice. Two-way Repeated Measures (RM) ANOVA. Statistic analysis details in Additional file 1. Supplementary Statistical Analysis. Median and 25th -75th percentile (“+”indicates outlier). k–m Effect of GCN2 inhibition on clinical score, motor phenotype, and weight gain. WT n = 4 mice, WT + GCN2ib n = 6 mice, G93A n = 6 mice, G93A + GCN2ib n = 6 mice. Two-way RM ANOVA and Tukey´s post-hoc test. Statistic analysis details in Additional file 1. Supplementary Statistical Analysis. *P < 0.05, **P < 0.01, **P < 0.001