Additional file 6 of Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity

Additional file 6. Figure S6: A four-fold increase in GA100 expression is not as toxic as expression of GA400 during development. A Q-RT-PCR quantification of GA transcript levels in heads of 2-day-old flies expressing one copy of GA100 (attP2), two copies of GA100 (2xGA100; attP40, attP2), three copies of GA100 (3xGA100; attP40, attP2/attP2) under control of the GMR-Gal4 driver; or two copies of both the GMR-Gal4 and the GA100 transgene (2xGMR-Gal4; 2xGA100 (attP2)). GA transcript levels were increased 2-fold between GA100 and 2xGA100, and 4-fold between GA100 and 3xGA100 or 2xGMR-Gal4; 2xGA100 (One-way ANOVA + Tukey’s multiple comparisons test; n = 4 replicates of 20 fly heads; **P < 0.01, ***P < 0.001, ***P < 0.0001). B Representative eye images of flies expressing GA100, 2xGA100, 3xGA100 or GA400 under the control of GMR-Gal4 and 2xGMR-Gal4, 2xGA100 and the corresponding control with two copies of the GMR-Gal4 (2xGMR-Gal4) driver. C Quantification of eye size area from B. Data were normalized to the eye size of GMR-Gal4/+ control flies. In contrast to GA400, which significantly reduced eye size, a 4-fold increase in GA100 levels in 3xGA100 or 2xGMR-Gal4; 2xGA100 flies did not affect eye size (One-way ANOVA + Tukey’s multiple comparisons test; n = 12 fly eyes per genotype; ***P < 0.0001). Note, 2xGMR-Gal4 flies showed a rough eye phenotype, which was not changed by co-expression of 2xGA100. D Egg-to-adult viability of flies expressing polyGA proteins under the control of the GMR-Gal4 driver. While GA400 expression significantly reduced egg-to-adult viability, expression of GA100 even at higher expression levels had no effect (One-way ANOVA + Tukey’s multiple comparisons test; n = 8-10 independent vials and 10-109 eggs/vial; ***P < 0.0001).