Extended Data Figure 9. iPS motor neurons from patients carrying the C9orf72 HRE show an increased number of P bodies and increased sensitivity to tunicamycin.

a) The number of processing bodies (P bodies), but not the size, is significantly increased in iPS motor neurons from patients carrying the C9orf72 HRE. There is an almost two-fold increase in the total number of P bodies per iPS neuron from C9orf72 HRE patients. The number and size of P bodies from 30 fields of view were quantified in two independent patient iPS motor neuron lines and two control lines. Data are means ± s.e.m. n = 43. *P < 0.05. b) The representative images of P bodies in iPS neurons were visualized and quantified using a DCP1A antibody (Abnova H00055802-M06, green), and the imaging and analysis are described in Supplementary Methods. c) The percent change in cell death with increasing doses of tunicamycin indicates that it takes lower concentrations of tunicamycin to induce significant toxicity in C9orf72 HRE iPS motor neurons than in control neurons. Moreover, there is a significant difference in tunicamycin responses at 0.3 μM when compared between C9orf72 HRE and C9orf72 WT iPS motor neurons. Each iPS line was differentiated twice and the tunicamycin sensitivity at varying concentrations (0.0, 0.1, 0.3, 0.6, 1.0 and 3.0 μM) was analyzed twice. The mean number of PIpositive iPS neurons was normalized to that of untreated iPS neurons from the same line. Data are means ± s.e.m. n = 69/58 (0.0), 62/69 (0.1), 75/55 (0.3), 54/49 (0.6), 55/52 (1.0), and 60/53 (3.0) for C9orf72 WT/HRE samples, respectively. **P < 0.01. d) Representative iPS motor neuron images showing the increased PI staining (white) in response to increasing tunicamycin concentrations (Supplementary Method).