Fig. 4

Presence of the C9orf72 HRE results in increased levels of insoluble full-length and C-terminal fragments of TDP-43 and an increase in p62 levels. a Schematic representation of the worklow used to separate soluble and insoluble fractions from iPSC-derived MNs. b Representative Western blot showing TDP-43 in total (unfractionated), soluble and insoluble fractions. c–g Quantifications of (b), measuring the levels of full-length TDP-43 in the total fraction (c), full-length TDP-43 in the soluble fraction (d), full-length TDP-43 in the insoluble fraction (e), the c-terminal fragment of 35 kDa (CTF-35) in the insoluble fraction (f) and the c-terminal fragment of 25 kDa (CTF-25) in the insoluble fraction (g). Each dot represents one biological replicate. h Immunocytochemistry (ICC) images of 40-day-old C9orf72 and isogenic control iPSC-derived MNs stained for the aggregate marker p62 and a neuronal marker TUJ1. Scale bar = 10 µm. i, j Quantifications of the corrected total cell fluorescence (CTCF) (i) and p62 puncta number per cell (j) from the ICC images shown in (h); each dot represents a confocal image containing one single MN that was analyzed (n = 60 for all conditions). k Representative Western blot detecting p62 in post-mortem human central cortex tissue lysates from C9orf72 ALS patients (C9orf72), ALS patients tested negative for the C9orf72 HRE (Non-C9 ALS) and healthy controls (Control). l, Quantification of (k), measuring p62 protein levels; each dot represents one tissue sample (C9orf72, n = 9; Non-C9 ALS, n = 8; Control, n = 7). GAPDH was used to normalize data. Data represent mean ± SEM; data are pooled from three independent differentiations. Statistical significance was assessed by one-way ANOVA and Tukey’s multiple comparison test (i, j) or Kruskal–Wallis test and Dunn’s multiple comparison test (c–g, l); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001