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. 2020 Dec 10;9:e59419. doi: 10.7554/eLife.59419

Figure 1. Autophagy receptor Ref(2)P/p62 genetically suppresses G4C2-HRE-mediated degeneration.

(A) 15-day-old Drosophila eyes expressing GMR-Gal4 +/- UAS-30R (GMR>30R) with RNAi background (control), ref(2)P RNAi#1 or overexpression (OE) of ref(2)P. (B) Quantification of external eye degeneration in A by semi-quantitative scoring system. Data are reported as mean ± SEM. Kruskal-Wallis test, p<0.0001, followed by Dunn’s multiple comparisons, n > 15 adults. (C) 15-day-old Drosophila eyes expressing GMR-Gal4 +/- UAS-36R (GMR >36R) along with UAS-luciferase RNAi (control), UAS-ref(2)P RNAi #1, UAS-ref(2)P RNAi #2, or UAS-ref(2)P OE. (D) Quantification of external eye degeneration in C by semi-quantitative scoring system. Data are reported as mean ± SEM. Kruskal-Wallis test, p<0.0001, followed by Dunn’s multiple comparisons, n = 23, 62, 28, 10 adults respectively. (E) Percent of pupal eclosion of adult flies expressing the motor neuron driver vGlut-Gal4 +/- UAS-30R and RNAi background control or UAS-ref(2)P RNAi #1. Fisher’s exact test, n > 100 pupa. (F) Percent of pupal eclosion of adult flies expressing the motor neuron driver vGlut-Gal4 +/- UAS-44R along with UAS-luciferase RNAi, UAS-ref(2)P RNAi #1, or UAS-ref(2)P OE. Fisher’s exact test, n > 55 pupa. (G) Adult Drosophila expressing UAS-30R under the control of the inducible, pan-neuronal elavGS induced with 200 µM RU486 or vehicle alone and co-expressing control or UAS-ref(2)P RNAi #1. Data are reported as mean ± SEM. One-way ANOVA, ****p<0.0001, with Sidak’s multiple comparisons test, n = 9, 8, 8, 8 groups of 10 flies.

Figure 1.

Figure 1—figure supplement 1. Ref(2)P/p62 genetically modifies G4C2-HRE.

Figure 1—figure supplement 1.

(A) GMR >30R expressing fly eyes at 15 days of age crossed to control (Canton S) or UAS-ref(2)P RNAi #2, demonstrating suppression of 30R-mediated eye degeneration with ref(2)P knockdown. (B) Levels of ref(2)P mRNA measured by quantitative RT-PCR in Drosophila expressing UAS-30R under the control of Act-Gal4 with UAS-ref(2)p RNAi #1 or UAS-ref(2)p RNAi #2. Data are presented as mean ± SEM. One-way ANOVA, p=0.0105 followed by Sidak’s multiple comparisons, n = 5 per genotype. (C) Levels of G4C2 mRNA measured by quantitative RT-PCR in flies expressing UAS-30R under the control of GMR-Gal4 either with or without UAS-ref(2)P RNAi. Data are presented as mean ± SEM. Student’s t-test, n = 3 per genotype. (D) GMR-Gal4 expressing an alternate codon UAS-poly(GR)36 alone (control) or co-expressed with UAS-ref(2)P RNAi #1 at 15 days of age.