Fig. 8. Expanded NOTCH2NLC polypeptides interact with and sequester hnRNPM while hnRNPM overexpression alleviates cellular toxicity.

(A) Interaction of NOTCH2NLC-polyG and endogenous hnRNPM in HEK293 cells transfected with NOTCH2NLC-(GGC)₁₇-GFP-HA or NOTCH2NLC-(GGC)₉₈-GFP-HA was determined by coimmunoprecipitation. (B and C) Shown are the images of immunofluorescence staining against hnRNPM and NOTCH2NLC-polyG (B) or NOTCH2NLC-polyA (C) in the hippocampus of NOTCH2NLC-(GGC)₁₇ and NOTCH2NLC-(GGC)₉₈ mice. Green, NOTCH2NLC-polyG/polyA; magenta, hnRNPM; blue, DAPI. Scale bars, 20 μm. (D) Shown are representative images of immunofluorescence staining against hnRNPM in hNPCs. Green, hnRNPM; magenta, ubiquitin; blue, DAPI. Scale bar, 20 μm. (E) Cell viability of Neuro-2a cells transfected with NOTCH2NLC-GFP plus control or hnRNPM constructs was analyzed by the CCK-8 method. Data are presented as means ± SEM. (N = 4 independent experiments, one-way ANOVA followed with Tukey’s multiple comparisons test was performed, *P = 0.0327). n.s., not significant. (F and G) Cell viability of cultured mouse primary cortical neurons transfected with NOTCH2NLC-GFP plus control or hnRNPM constructs. Data are presented as means ± SEM (N = 4 independent experiments, at least 120 cells were counted per experiment, one-way ANOVA followed with Tukey’s multiple comparisons test was performed, ***P < 0.0005, ****P < 0.0001). (F) Representative immunofluorescence image. Green, GFP; magenta, Tuj1; gray, His. Scale bar, 10 μm.