Extended Data Figure 8. Patients carrying the C9orf72 HRE show phenotypes indicative of nucleolar stress.

a) Another nucleolar component, nucleophosmin/B23 (green), shows a dispersed localization as seen for NCL in B lymphocytes from C9orf72 HRE patients. B23 (mouse monoclonal, B0556, Sigma) IF staining followed the manufacturer’s recommendations and the protocol described in Methods. B lymphocytes images were obtained as described in Methods. b) There is a significant increase in the total nuclear area occupied by B23 in B lymphocytes from a patient carrying the C9orf72 HRE when quantified similarly to NCL (Extended Data Figure 7a). The data are means ± s.e.m. n = 38 and n = 49 for representative C9orf72 WT and C9orf72 HRE B lymphocytes, respectively. *P < 0.05. c) The processing of the 45S pre-rRNA into the mature 28S, 18S, and 5.8S rRNAs that occurs in the nucleolus is depicted. d) Maturation of the 45S pre-rRNA to the 28S, 18S, and 5.8S rRNAs is impaired in patients carrying the C9orf72 HRE. B lymphocytes from patients (n = 6) show a decrease in 45S rRNA processing, but the processing is significantly decreased in the motor cortex tissues of patients carrying the C9orf72 HRE relative to controls (n = 4, 3, and 6 for C9orf72 WT, non-C9orf72 ALS, and C9orf72 HRE, respectively). Furthermore, ALS patients that do not carry the C9orf72 HRE show no significant impairment in 45s maturation (non-C9orf72 HRE ALS, n = 3). The primers for 45S, 28S, 18S, and 5.8S rRNAs (Extended Data Table 1) were previously described⁴⁷. The linear-fold change in mature rRNA levels was normalized relative to the parent 45S rRNA levels. Data are means ± s.e.m. *P < 0.05.