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. 2015 May 7;24(15):4317–4326. doi: 10.1093/hmg/ddv165

Figure 4.

Figure 4.

CGG repeats elicit UPS impairment via FMRpolyG translation. (A) Representative fluorescent (top) and DIC (bottom) images from HeLa cells that stably express the UPS reporter G76V-Ub-GFP. Cells were transfected with an empty vector or a vector expressing Ataxin 3 with 80 glutamines, Q80, or were treated for 24 h with 10 µm lactacystin, an inhibitor of the UPS. (B) Schematic of cell-based constructs. (C) Representative images of G76V-Ub-GFP HeLa cells transfected with the indicated constructs and imaged by confocal microscopy. (D) Quantitation of the percentage of red cells (indicating transfection) where green fluorescence was present at 48 h post -transfection. *P < 0.05 compared with mCherry alone. **P < 0.05 compared with the indicated genotype. (E) qRT-PCR quantifying RNA expression from each of the constructs. (F) Western blot to mCherry in cells either treated with lactacystin or transfected with the indicated constructs. MCherry runs at ∼25 kD (indicated by *). Higher-molecular-weight bands represent RAN translation product through the CGG repeat (indicated by <) or AUG-driven production of FMRpolyG (indicated by #).