Fig. 6

C9orf72 KO MNs do not show with defects in lysosomal trafficking or TDP-43 localisation but do present with a reduction in cell viability. A–c, Quantification of vesicle transport along neurites in control and C9 KO iPSC-derived MNs loaded with Lysotracker Red. Total number of tracks per 100 particles (a), total displacement per 100 particles (b) and average speed of particles (c) were analyzed. Each dot represents a well that was analyzed (CTRL, n = 57; C9 KO, n = 63). d Immunocytochemistry (ICC) of aged, 60-day-old control and C9 KO 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. e, f Quantifications of the nuclear vs cytoplasmic (N/C) TDP-43 ratio (e) and the corrected total cell fluorescence (CTCF) of pTDP-43 (f) from the ICC images shown in (d). Each dot represents a cell that was measured (CTRL, n = 153; C9 KO, n = 153). g Quantification of the relative cell viability in 60-day-old C9 KO MNs relative to its isogenic control line 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 and the average intensity of the C9 KO line was normalized to that of its isogenic control line; each dot represents one biological replicate. h 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 171 and 410 TUJ1-positive cells were scored for cleaved caspase-3 staining. Data represent mean ± SEM; data are pooled from five independent differentiations. Statistical significance was assessed by unpaired t-test (a–c, e, f, h) or one sample t-test (g); *p < 0.05, **p < 0.01