Fig. 4

a Control UAS-(G4C2)8 and expanded UAS-(G4C2)49 transgenes were expressed in the adult fly nervous system using the drug-inducible Gal4 driver, elavGS, for 16d. Quantitative PCR (qPCR) analysis of endogenous dHSF1 and HSF1-regulated genes revealed significant upregulation with (G4C2)49 expression compared to (G4C2)8 controls. Differences in expression are likely underestimated as the analyses include neuronal and non-neuronal tissue while (G4C2)n was expressed only in neurons. b qPCR analysis of a dHSF1 overexpression mutant fly line shows endogenous HSF is upregulated approximately 2-fold in mutant flies compared to control. c Western immunoblot analysis of expression of a control UAS-LacZ transgene confirmed that the HSF OE mutant did not affect the Gal4/UAS expression system. d (G4C2)49 was expressed in the optic system of control animals or HSF OE animals using Gmr-Gal4. (G4C2)49 causes toxicity seen by pigment loss, reduced eye size, and disruptions in the normal ommatidial organization. In HSF OE animals, toxicity of (G4C2)49 is enhanced – animals have increased pigment loss, increased disruption of ommatidial organization, and further reduced eye size. Expression of control (G4C2)8 in the fly optic system (Gmr-Gal4) of control and HSF OE animals does not affect the external eye. e Quantification of the external eye degenerative phenotype caused by (G4C2)49 expression shows enhancement in HSF OE animals versus control animals to be consistent and statistically significant (n = 6). Animals received a score between 0 (WT eye) and 8 (lethality caused by extreme degeneration in the optic system). (G4C2)49 expression causes an average score of 4 in controls. f Gmr-GAL4 driven expression of (GR)36 shows toxicity in control scenarios like (G4C2)49. HSF OE in these animals also causes enhanced toxicity (increased pigment loss, increased disruption of ommatidial organization, and reduced eye size) g Quantification of the external eye degenerative phenotype caused by (GR)36 expression shows enhancement in HSF OE animals versus control animals to be consistent and statistically significant (n = 7). Animals received a score between 0 (WT eye) and 11 (lethality caused by extreme degeneration in the optic system) while (GR)36 causes an average score of 5 in controls. (All plots: mean +/− SD, unpaired, student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)