Fig. 2. Translated AAGGG repeat products are detected in brains of patients with CANVAS.

(A) Schematic of potential peptide products from sense and antisense strand of the repeat expansion locus. (B) Quantification of foci positive neurons for control (n = 3) and CANVAS (n = 3) patient iPSC-derived neurons (1100 to 2000 cells per group per probe). n = 2 biological replicates from three independent patient-derived cell lines. Representative confocal images are shown in fig. S2B. (C) Immunoblot from HEK293 cells expressing plasmids encoding intronic sense or antisense AAGGG/CCCTT repeat reporters in the +0/+1/+2 reading frames (left) and Nano-luciferase expression assay quantification (right). n = 7 biological replicates. Data were analyzed by one-way ANOVA with Sidak’s post hoc multiple comparison tests. (D) ICC of HEK293 cells transfected with plasmids encoding intronic sense or antisense AAGGG/CCCTT repeat reporters with C-terminal triple tags in the +0/+1/+2 reading frames. (E) Expression analysis of lysates from HEK293 cells transfected with control plasmid +2 Sense (AAGGG)₆₁ plasmid using anti-FLAG M2 (1:1000) and anti-KGREG (1:100) antibodies (left). (F) Left: IHC of control and RFC1 expansion CANVAS patient postmortem cerebellar vermis tissue stained with sense anti-KGREG antibody (1:100, acid AR). Scale bars, 500 μm (4×), 50 μm (60×), and 20 μm (inset). Right: Rater blinded quantification of all 20 postmortem tissues (tissue images and quantification for each sample in fig. S4). (G) Cumulative hazard plot for rat cortical neurons expressing CGG100 (positive control) or CANVAS intronic expression plasmids containing 61 repeats of the indicated type over 10 days. Results from eight technical replicates/three biological replicates; n = numbers of cells assessed per condition. ns, not significant. *hazard ratio = 1.339, P = 0.025, Cox proportional hazards analysis.