Additional file 8 of Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity

Additional file 8. Figure S8: C-terminal GFP and mCherry protein tags abrogate GA400 toxicity. A Egg-to-adult viability, B representative eye images and C eye size area quantification of female flies expressing untagged GA400, GA400-GFP or GA400-mCherry under the control of the GMR-Gal4 driver. A Expression of GA400, but not that of GA400-GFP or GA400-mCherry, significantly reduced egg-to-adult viability (One-way ANOVA + Tukey’s multiple comparisons test; n = 10 independent vials and 4-127 eggs/vial; ***P < 0.0001). #1-3 indicate independently generated transgenic fly lines for GA400-GFP and GA400-mCherry. B-C GA400 expression caused a severe reduction in eye size compared to GMR-Gal4/+ flies (One-way ANOVA + Tukey’s multiple comparisons test; n = 10 fly eyes; ***P < 0.0001), while expression of GA400-GFP or GA400-mCherry did not affect eye size. Eye size area is shown relative to GMR-Gal4/+ control flies. D Survival curves of flies expressing GA400, GA400-GFP, GA400-mCherry under control of the elav-GS driver. GA400, but not GA400-GFP or GA400-mCherry expression, significantly shortened survival compared to elav-GS control flies (GA400 vs elav-GS; log-rank + Bonferroni’s multiple corrections test; n = 150 female flies per genotype; ***P < 0.0001). E Western blot analysis of fly heads expressing GA400, GA400-GFP or GA400-mCherry under the control of elav-GS for 5 days. Blots were probed with an anti-GA antibody. F-H Quantification of the western blot from E. F Total GA levels were similar between GA400 and GA400-mCherry, and increased in GA400-GFP-expressing flies. G HMW GA levels were increased and H LMW GA levels were decreased in GA400 heads compared to those in GA400-GFP or GA400-mCherry extracts. Statistics in A, C, F-I: **P < 0.01, ***P < 0.001, ***P < 0.0001; One-way ANOVA + Tukey’s multiple comparisons test; F-I: n = 4 replicates of 20 fly heads.