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. 2026 Aug 6;23(5):e00970. doi: 10.1016/j.neurot.2026.e00970

Fig. 2.

Fig. 2

C9orf72 overexpression promotes aggregates and cell death in mHTT-expressing cells. (a–h) N2a cells were co-transfected with GFP-mHTT and either C9orf72 or an empty control vector, and analyzed 48 h post-transfection by fluorescence microscopy and immunoblotting. (a) Schematic diagrams of the plasmids used for transfection, including GFP-mHTT (HD exon 1 containing 84 CAG repeats) and long isoform C9orf72 (C9 plasmid) with a C-terminal HA tag. Co-transfection ratio of GFP-mHTT and C9orf72 plasmid was 1:1. (b) Representative fluorescence images of N2a cells expressing GFP-mHTT with either control vector or C9orf72. Hoechst staining marks nuclei (blue), and GFP fluorescence indicates GFP-mHTT (green). Increased GFP-positive aggregates are observed in cells expressing C9orf72. Scale bar = 200 μm. (c) Representative immunoblots showing insoluble GFP-mHTT (stacking gel), soluble GFP-mHTT, C9orf72 and γ-tubulin. (d) Quantification of insoluble GFP-mHTT levels normalized to γ-tubulin, showing increased aggregation upon C9orf72 overexpression. (n = 5). (e) Representative immunoblots showing C9orf72, caspase-3, cleaved caspase-3, and γ-tubulin. (f) Quantification of cleaved caspase-3 normalized to γ-tubulin, indicating increased apoptotic signaling in cells overexpressing C9orf72. (n = 5). (g) Representative fluorescence images of N2a cells stained with Hoechst (blue) and propidium iodide (PI, red). Increased PI-positive cells are observed in the C9orf72-overexpressing group. Scale bar = 100 μm. (h) Quantification of cell survival rate based on PI staining, showing reduced viability in C9orf72-overexpressing cells compared with control. (n = 6–7). (i–k) Primary neurons were infected with GFP-mHTT together with either control vector or C9orf72 and analyzed by immunoblotting. (i) Representative immunoblots of primary neurons expressing GFP-mHTT with either control vector or C9orf72. (j) Quantification of insoluble GFP-mHTT normalized to γ-tubulin. (k) Quantification of cleaved caspase-3 normalized to γ-tubulin. (n = 6). Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by unpaired t-test.