Fig. 4: C9 ALI-COs show ALS/FTD-related pathological hallmarks at 150 DIV.
a, Dot blot shows anti-poly(GA) immunoreactivity in C9 ALI-COs (n = 3 independent ALI-COs per group). b,c, Representative WB images for the ER stress/UPR elements p-EIF2α/EIF2α, the stress granule marker PABP1, the autophagy marker P62, and β-actin in ALI-CO samples from experiments quantified in d. d, Quantification of WB band densities normalized to β-actin. Left graphs: data represent the mean ± s.e.m.; for p-EIF2α/EIF2α ratios and PABP1 n = 20 control (H-L1, H-L2, ISO-L2) and n = 16 C9 (C9-L1, C9-L2) and for P62 n = 23 control (H-L1, H-L2, ISO-L2), n = 22 C9 (C9-L1, C9-L2) ALI-COs, respectively; two-tailed unpaired t-test. Right graphs: data represent the mean ± s.e.m. from experiments comparing C9-L2 to its isogenic mutation-corrected ISO-L2 control and non-related ALI-COs; n = 4 independent ALI-COs per group; one-way ANOVA with Fisher’s LSD test. e, Representative confocal microscopy image stacks (left) and quantification (right) of ALI-COs with single plane x–z orthogonal views, showing P62 immunolabeling in GFAP+ astroglia and MAP2+ neurons with DAPI+ nuclei and quantification of P62+ area overlaps with either GFAP+ or MAP+ territories. Data are expressed as the mean percentage ± s.e.m.; n = 7 independent ALI-COs per group; two-tailed unpaired t-test. f, WB image (upper) of ALI-COs, corresponding to samples in c, showing autophagy marker LC3 immunolabeling with bands representing LC3-I early, LC3-II late autophagosomes and β-actin. Lower left: data in the graph represent the mean ± s.e.m. of LC3-II/LC3-I ratios; n = 20 control (H-L1, H-L2, ISO-L2), n = 16 C9 (C9-L1, C9-L2) independent ALI-COs per group. Lower right: data in the graph represent the mean ± s.e.m. from experiments comparing C9-L2 to ISO-L2 control and non-related ALI-COs; n = 4 independent ALI-COs; one-way ANOVA with Fisher’s LSD test. Scale bar, 10 μm (e). See Supplementary Table 3 for detailed statistics and source data for unprocessed WB images.
