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Fig. 4. Transgenic zebrafish expressing human ataxin-3 with 84 glutamines develop ataxin-3+ protein aggregates. (A) Confocal microscopy was performed on 6-day-old zebrafish larvae expressing EGFP-tagged human ataxin-3 containing a short polyglutamine stretch (28Q) or long polyglutamine stretch (84Q). (B) Manual counting of EGFP aggregates revealed that significantly more aggregates were present in 6-dpf zebrafish expressing EGFP ataxin-3 84Q than those expressing EGFP ataxin-3 23Q. (C) Quantification of the percentage of cells harbouring protein aggregates revealed that a greater number of cells were affected with aggregates in zebrafish expressing EGFP ataxin-3 84Q. (D) Measurement of the size of protein aggregates observed via confocal microscopy revealed that zebrafish expressing EGFP ataxin-3 84Q also display aggregates that are significantly larger in size compared to the aggregates found in zebrafish expressing EGFP ataxin-3 23Q. (E) Western blotting was used to examine the relative expression of human ataxin-3 across the examined genotypes. (F) Densiometric analysis revealed significantly greater expression of EGFP-fused human ataxin-3 in transgenic zebrafish compared to non-transgenic siblings. Data are meanąs.e.m. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey's post-hoc test or Student's t-test, paired, two-tailed). n=4-5 experimental replicates.