PR toxicity increases nuclear accumulation of Staufen in neurons. (A) Quantification of nuclear RBPs in C4da neurons expressing V5-PR36 compared to controls. The pixel number and the mean intensity of nuclear RBPs were multiplied to provide an estimate of the amount of nuclear RBPs. ^**P = 0.0026, ^****P < 1.0|$\times$|10⁻⁴ by two-tailed student’s t-test; error bars, ± SEM; n ≥ 13 RBP puncta; n = 3 larvae. (B) Subcellular localization of Staufen-GFP in larval C4da neurons of controls (CTRL) or expressing V5-PR36 (Genotypes. CTRL: UAS-stau-GFP/+; PPK^1a-gal4 > UAS-mCD8RFP/+, V5-PR36: UAS-stau-GFP/+; PPK^1a-gal4 > UAS-mCD8RFP/UAS-V5-PR36). Merge: merged with plasma membrane marker. Bottom right insets are magnified images of the nucleus. Dashed circular lines (white) outline the nuclei. Arrow (white) indicates increased nuclear Staufen-GFP puncta. Scale bars (yellow), 2 μm; (white), 10 μm. (C) Individual nuclear Staufen-GFP puncta in C4da neurons with denoted genotypes were counted and plotted. y-axis: the mean intensity of Staufen-GFP puncta in the nucleus; x-axis: the pixel number of Staufen-GFP puncta in the nucleus. n ≥ 19 Staufen-GFP puncta; n = 3 larvae. (D) Western blot showing the expression level of Staufen-GFP in fly heads (Genotypes. stau-GFP + CTRL: UAS-stau-GFP; elavGS-gal4 > UAS-pACU2-empty, stau-GFP + V5-PR36: UAS-stau-GFP; elavGS-gal4 > UAS-V5-PR36). α-tubulin was used as a loading control. The transgenic expression was induced by feeding 100 μM of RU486 for 10 days after eclosion at 27°C. (E) Quantification of the amount of Staufen-GFP normalized to the control. ^**P = 0.0024 by paired two-tailed student’s t-test; error bars, ± SEM; n = 3 with 2 technical repeats. (F) Western blot showing the expression level of endogenous Staufen in fly heads (Genotypes. w¹¹¹⁸: +/+, V5-PR36: +; elavGS-gal4 > UAS-V5-PR36). α-tubulin was used as a loading control. The transgenic expression was induced by feeding 100 μM of RU486 for 10 days after eclosion at 27°C. (G) Quantification of the amount of Staufen-GFP normalized to the control. ns: not significant by paired two-tailed student’s t-test; error bars, ± SEM; n = 3 with 2 technical repeats. (H) Images of C4da neurons expressing either one or two copies of stau-GFP. Merge: merged with plasma membrane marker. Scale bar (white), 10 μm. (I) Quantification of the amount of nuclear Staufen-GFP normalized to the control. ns: not significant by two-tailed student’s t-test; error bars, ± SEM; n ≥ 58 Staufen-GFP puncta; n = 3 larvae. (J) IHC of dissected third instar larval motor neurons shows that V5-PR36 expression increased nuclear accumulation of Staufen-GFP (Genotypes. CTRL: UAS-stau-GFP/+; D42-Gal4 > UAS-mCD8RFP/+, V5-PR36: UAS-stau-GFP/+; D42-Gal4 > UAS-mCD8RFP/UAS-V5-PR36). Dashed circular lines (white) outline the nuclei. Arrows (white) indicate stronger nuclear Staufen-GFP signal compared to the control. Scale bar (white), 5 μm.