a) Quantification of nucleolin (NCL) in the nucleus shows significant NCL nuclear dispersion in B lymphocytes, iPS motor neurons, and fibroblasts from C9orf72 HRE patients. A single focal plane was obtained through the center of the nucleus (Hoechst staining; blue). To quantify the area differences in NCL relative to the size of the nucleus a single lower threshold setting in ImageJ (NIH), ranging from 25–75 (which provided quantification of both dispersed NCL and dense nucleolar NCL), was used to measure the pixel area of NCL relative to the area of the nucleus outlined by the Hoechst staining (depicted in the cartoon). The measurements for the C9orf72 HRE cells were then normalized to those for controls (Prism). Data are means ± s.e.m. n = 110 and n = 112 for 3 cell lines examined for the C9orf72 WT and C9orf72 HRE B lymphocytes, respectively. n = 29 for iPS motor neurons from 2 C9orf72 HRE patients and for the iPS motor neuron controls. A representative fibroblast line for C9orf72 WT, SOD1 D90A ALS, and C9orf72 HRE was quantified. n = 21. *P < 0.05; **P < 0.01; ****P < 0.0001. b) There are no significant changes in NCL protein levels between patients with and without the C9orf72 HRE, despite its mislocalization. The Western blotting analysis was performed as described in the Methods, following a typical protocol with the B lymphocyte lysates from patients and controls. The bands were visualized using a quantitative LI-COR imager, and the intensities for NCL and tubulin protein were quantified using Odyssey software. NCL levels were normalized against those of tubulin. Data are means ± s.e.m. n = 6. c) RNA foci are a unique feature of the C9orf72 HRE ALS pathology. Motor cortex tissue from C9orf72 WT (non-ALS) and non-C9orf72 sALS show no RNA foci or NCL colocalization, which is observed in motor cortex tissue from patients carrying the C9orf72 HRE (). All images were obtained as described in Methods. d) The protein hnRNP F, K, and U, identified from the pulldown ( and ), show no phenotypic differences in localization between patients carrying the C9orf72 HRE and controls, as previously observed in iPS motor neurons from FTD patients. n = 3. e) A representative HEK293T cell transfected with the control transcript shows the typical NCL localization (green), in contrast to the nucleolar dispersion observed in cells transfected with (GGGGCC) (). f) NCL is significantly mislocalized when HEK293T cells are transfected with (GGGGCC) transcripts, as compared to (GGGGCC)3 or control transcripts. RNA transfections were performed in duplicate, except for No RNA and (GGGGCC)3. Data are means ± s.e.m. n = 27, 50, 37, and 60 for the No RNA, CTL RNA, (GGGGCC)3, and (GGGGCC)21, respectively. ****P < 0.0001. g) There is a significantly decreased cell viability in HEK293T cells transfected with the (GGGGCC)21 abortive RNA transcripts when compared to a control RNA transcript. Cytotoxicity measurements were performed in a 96-well plate with HEK293T cells that had been transfected with 0–500 ng/mL of control RNA or (GGGGCC)21 repeat-containing RNAs generated from the in vitro transcription as described earlier and presented in . Cell viability was measured using the Cytotox Glo kit (Promega), with the fluorescence (viable cells) and luminescence (dead) measured on a Synergy H1 microplate reader. Data are means ± s.d. n = 3. *P < 0.05.