Fig. 5

The autophagic pathway is impaired in C9orf72 MNs. a, Representative Western blot of iPSC-derived MNs treated with either DMSO, the autophagy inhibitor Bafilomycin A1 (Baf A1), the autophagy inducer Torin 1 or a combination of the latter, immunostained for LC3-I and LC3-II, p62, LAMP1 and β-actin, used to measure levels of autophagosomes, aggregates, lysosomes or for protein normalization respectively. b,c,d, Quantifications of the Western blot shown in (a), measuring the relative levels of p62 (b), the ratio of LC3-II/LC3-I (c) and LAMP1 (d); each dot represents one biological replicate. e Schematic representation of the autophagic flux assay. By inhibiting the autophagy pathway with high doses of Baf A1 and measuring the concomitant increase in LC3-II or p62, we get an estimate of the amount of LC3-II or p62 that would have been degraded when no drugs were added (= autophagic flux). f, g Quantifications of the Western blot shown in (a), measuring the levels of p62 (f) or LC3-II (g) after treatment with Baf A1 and dividing those with the levels of either p62 or LC3-II without treatment; each dot represents one biological replicate. h,i, Quantifications of the Western blot shown in (a), measuring the relative levels op p62 (h), LC3-II (i) after treatment with Torin 1 and dividing those with the levels of either p62 or LC3-II without treatment; each dot represents one biological replicate. j Experimental outline of the Baf A1 vulnerability assay in iPSC-derived MNs. Following normal MN differentiation, DMSO, 1 µM or 3 µM of Baf A1 are added to the medium at day 37, 38 or 39. At day 40, the CellTiter-Glo® assay was used to asses cell survival. k, l, m, Quantification of the survival rate after 24 h (k), 48 h (l) or 72 h (m) treatment with Baf A1. The survival rate of iPSC-derived MNs is calculated by normalizing the CellTiter-Glo® signal of Baf A1-treated cells to DMSO control-treated wells; each dot represents the average of one biological replicate. Data represent mean ± SEM; data are pooled from four-five (k–m) or eight-nine (b–d, f–i) independent differentiations. Statistical significance was assessed by one-way ANOVA and Tukey’s multiple comparison test (b–d, f–i, k–m); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant