Figure 5.
Autophagy is induced by blocking PERK in Rh1P37Hphotoreceptor cells. (A) Proteomic profiling comparing protein levels in retinas of Rh1P37H-GFP perkRNAi (GMR-Gal4/UAS-perkRNAiRh1P37H-GFP) and Rh1P37H-GFP flies (Tandem Mass Tag-LC MS/MS assay). A total of 1,339 proteins were confidently identified (at least two unique peptides per protein). A subset of proteins up- or downregulated in Rh1P37H-GFP perkRNAi cells are highlighted by protein identification. (B and C) Western blotting confirmed that Ref(2)P/P62 was upregulated in Rh1P37H-GFP perkRNAi flies. This was abolished by expression of ire1RNAi and xbp1RNAi. Ref(2)P/P62 protein is indicated by the red arrow. Error bars indicate SEM (n = 6); ns, not significant, **P < 0.01, ***P < 0.001 (one-way ANOVA, Sidak’s multiple comparisons test). (D) Tangential views of retina expressing Rh1P37H-GFP (green) and Rh1-RFP (red) with perkRNAi and/or ire1RNAi/xbp1RNAi staining against Ref(2)P/P62 (blue). Scale bar, 20 μm. (E) qPCR analysis of ref(2)P/p62 mRNA levels in Rh1P37H-GFP perkRNAi and Rh1P37H-GFP perkRNAiire1RNAi retinas compared with Rh1P37H-GFP controls. Error bars indicate SEM (n = 3); *P < 0.1, **P < 0.01 (one-way ANOVA, Sidak’s multiple comparisons test). (F) qPCR analysis showed that mRNA levels of autophagy-related genes (including atg1, atg2, atg3, atg8a, atg9, and atg18a) were upregulated in the retina of Rh1P37H-GFP perkRNAi flies, compared with Rh1P37H-GFP and Rh1P37H-GFP perkRNAiire1RNAi retina. Error bars indicate SEM (n = 3); *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two -way ANOVA, Sidak’s multiple comparisons test). 1-d-old flies of indicated genotypes were used. (G) qPCR analysis showed that mRNA levels of the ER-associated E3 ligase, hrd1 (bot not sordd1) was upregulated in the retina of Rh1P37H-GFP perkRNAi flies, compared with Rh1P37H-GFP and Rh1P37H-GFP perkRNAiire1RNAi retina. Error bars indicate SEM (n = 3); ns, not significant, ****P < 0.0001 (two-way ANOVA, Sidak’s multiple comparisons test). 1-d-old flies of indicated genotypes were used. Source data are available for this figure: SourceData F5.

Autophagy is induced by blocking PERK in Rh1 P37H photoreceptor cells. (A) Proteomic profiling comparing protein levels in retinas of Rh1P37H-GFP perkRNAi (GMR-Gal4/UAS-perkRNAiRh1P37H-GFP) and Rh1P37H-GFP flies (Tandem Mass Tag-LC MS/MS assay). A total of 1,339 proteins were confidently identified (at least two unique peptides per protein). A subset of proteins up- or downregulated in Rh1P37H-GFP perkRNAi cells are highlighted by protein identification. (B and C) Western blotting confirmed that Ref(2)P/P62 was upregulated in Rh1P37H-GFP perkRNAi flies. This was abolished by expression of ire1RNAi and xbp1RNAi. Ref(2)P/P62 protein is indicated by the red arrow. Error bars indicate SEM (n = 6); ns, not significant, **P < 0.01, ***P < 0.001 (one-way ANOVA, Sidak’s multiple comparisons test). (D) Tangential views of retina expressing Rh1P37H-GFP (green) and Rh1-RFP (red) with perkRNAi and/or ire1RNAi/xbp1RNAi staining against Ref(2)P/P62 (blue). Scale bar, 20 μm. (E) qPCR analysis of ref(2)P/p62 mRNA levels in Rh1P37H-GFP perkRNAi and Rh1P37H-GFP perkRNAiire1RNAi retinas compared with Rh1P37H-GFP controls. Error bars indicate SEM (n = 3); *P < 0.1, **P < 0.01 (one-way ANOVA, Sidak’s multiple comparisons test). (F) qPCR analysis showed that mRNA levels of autophagy-related genes (including atg1, atg2, atg3, atg8a, atg9, and atg18a) were upregulated in the retina of Rh1P37H-GFP perkRNAi flies, compared with Rh1P37H-GFP and Rh1P37H-GFP perkRNAiire1RNAi retina. Error bars indicate SEM (n = 3); *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two -way ANOVA, Sidak’s multiple comparisons test). 1-d-old flies of indicated genotypes were used. (G) qPCR analysis showed that mRNA levels of the ER-associated E3 ligase, hrd1 (bot not sordd1) was upregulated in the retina of Rh1P37H-GFP perkRNAi flies, compared with Rh1P37H-GFP and Rh1P37H-GFP perkRNAiire1RNAi retina. Error bars indicate SEM (n = 3); ns, not significant, ****P < 0.0001 (two-way ANOVA, Sidak’s multiple comparisons test). 1-d-old flies of indicated genotypes were used. Source data are available for this figure: SourceData F5.

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