Fig. 2

Effect of NF-κB transcription factor Relish in astrocytes on SCA3^polyQ78-induced degeneration. a Quantification of Relish or Dif/Dorsal activation in the head upon eye-specific expression of SCA3^polyQ78. Heads of control flies or flies expressing SCA3^polyQ78 in the eyes were analyzed for expression of Relish target genes (CecA, DptA) or Dif/Dorsal target genes (IM-1, IM-2). b Effect of modulation of Relish expression in astrocytes on SCA3^polyQ78-induced eye degeneration. Representative images of fly eyes expressing SCA3^polyQ78 (−) or SCA3^polyQ78 together with either astrocyte-specific expression of two independent RNAi constructs targeting Relish (Relish RNAi #1 and Relish RNAi #2), a Relish overexpression construct (Relish overexpression) or flies heterozygous for Relish (Relish −/+). c Quantification of the fraction of degeneration of the eyes shown in b. In these and other eye quantification experiments, at least 80 eyes of females were counted per genotype. Data are representative of at least three independent experiments ± SEM. d The effect of modulation of Relish expression in astrocytes on GFP fluorescence in SCA3^polyQ78-expressing eyes together with eye-specific mCD8-GFP and on the localization of astrocytes (expressing myr-RFP). Representative images of control eyes, eyes expressing mCD8-GFP, and astrocyte-specific myr-RFP (RFP) or together with coexpression of SCA3^polyQ78 in the absence or presence of Relish RNAi constructs (Relish RNAi #1 and Relish RNAi #2) or Relish overexpression in astrocytes. e Quantification of the effect of Relish signaling in astrocytes on SCA3^polyQ78-induced degeneration by using eye-specific expression of mCD8-GFP. Levels of mCD8-GFP were determined in lysates of fly heads of flies expressing SCA3^polyQ78 together with mCD8-GFP in the eyes and the effect of astrocyte-specific expression of Relish RNAi constructs (Relish RNAi #1 and Relish RNAi #2) in astrocytes, heterozygosity for Relish (Relish −/+), or overexpression of Relish in astrocytes was analyzed. Tubulin was used as a control for equal loading. Quantification of western blots is of at least three independent experiments. f Analysis of myr-RFP expression in astrocytes in lysates of control flies or flies expressing myr-RFP in astrocytes together with eye-specific SCA3^polyQ78 in the presence or absence of astrocyte-targeted Relish RNAi (Relish RNAi #1 or #2) or Relish overexpression. Quantification of western blots in two independent experiments. Genotypes: a control, GMR-QF2/+. SCA3^polyQ78, GMR-QF2/+; QUAS- SCA3^polyQ78/+. b, c -, GMR-QF2/+; QUAS-SCA3^polyQ78:: alrm-Gal4/+. Relish RNAi #1, GMR-QF2/+; QUAS-SCA3^polyQ78:: alrm-Gal4/UAS-Relish RNAi #1. Relish RNAi #2, GMR-QF2/+; QUAS-SCA3^polyQ78:: alrm-Gal4/UAS-Relish RNAi #2. Relish overexpression, GMR-QF2/+; QUAS-SCA3^polyQ78:: alrm-Gal4/UAS-GFP-Relish; Relish−/+, GMR-QF2/+; QUAS-SCA3^polyQ78:: alrm-Gal4; Relish E20/+. d–f As in b, but with additional eye-specific expression of QUAS-CD8-GFP or astrocyte-specific expression of UAS-myr-RFP