Fig. 3

Upon aging, C9orf72 MNs display mislocalization of TDP-43, increased phosphorylation of TDP-43 and a reduced cell viability. a Immunocytochemistry (ICC) of aged, 60-day-old C9orf72 and isogenic control MNs stained for TDP-43 and phosphorylated TDP-43 (pTDP-43). The presence of a typical neuronal morphology (which includes a soma and elongated neurites was used to select the MNs for analysis. Scale bar = 10 µm. b, c Quantifications of the nuclear vs cytoplasmic (N/C) TDP-43 ratio (b) and the corrected total cell fluorescence (CTCF) of pTDP-43 (c) from the ICC images shown in (a). Each dot represents a cell that was measured (C9-1, n = 124; C9-1iso, n = 145; C9-2, n = 133; C9-2iso, n = 132). d Schematic overview of the two methods used to measure cell viability. e Quantification of the relative cell viability in 60-day-old C9orf72 MNs relative to their isogenic controls as measured by the CellTiter-Glo® assay. For each independent differentiation, at least 6 technical replicates of 5000 cells were plated in 96-well plates the average intensity of each C9orf72 patient line was normalized to that of their respective isogenic control; each dot represents one biological replicate. f Quantification of the percentage of apoptotic cells staining positive for cleaved caspase-3 in the TUJ1-positive 60-day-old MN population. Each dot represents one biological replicate in which between 126 and 239 TUJ1-positive cells were scored for cleaved caspase-3 staining. Data represent mean ± SEM; data are pooled from four-five independent differentiations. Statistical significance was assessed by one-way ANOVA and Tukey’s multiple comparison test (e, f) or Kruskal–Wallis test and Dunn’s multiple comparison test (b, c); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001