Fig. 3: Loss of Aln causes dysregulated proteostasis.

a–d Micrographs of cryosection of adult eyes of w¹¹¹⁸ (a, b) or aln² (c, d) flies treated for 3 days at LD or 1 day at LL and stained for the PERK activity reporter ATF4-5′UTR-DsRed. Quantified in (e), bar graphs show number of heads and median with interquartile range, Scale bar in a: 50 µm and is the same for (a–d). f, g Quantification of retinal staining for BiP (f) or the IREI activity reporter XBP1-GFP (g). Example images in Supplementary Fig. 4a–h. Flies were treated for 3 days at LD or 1 day at LL. Bar graphs show number of separate retinas pooled from three independent experiments and median with interquartile range. e–g P-values from Kruskal–Wallis test followed by Dunn’s multiple comparison test are (e) WT,LD-LL: 0.014; aln², LD-LL:0.99; LD, WT-aln²: <0.0001; (f) WT,LD-LL: 0.029; aln², LD-LL:0.14; LD, WT-aln²: 0.0067; (g) WT,LD-LL: 0.023; aln², LD-LL:0.99; LD, WT-aln²: 0.017. Significance threshold: p < 0.05; **p < 0.01, ****p < 0.0001. h–k aln² retinas and brains display elevated autophagic flux, w¹¹¹⁸ (h, j) or aln² (i, k) flies stained for ATG8. Flies were treated without (h, i) or with CQ-containing food (j, k) to inhibit lysosomal degradation. Scale bar in (k) is 100 µm and is same for (h–k). l Bar graphs show quantification of Atg8 punctae in the w¹¹¹⁸ and aln² brains. Statistics: Kruskal–Wallis test followed by Dunn’s multiple comparison test. Bar graphs show number of separate retinas pooled from three independent experiments and median with interquartile range, *p < 0.05; **p < 0.01. m–p Cryosection of adult heads of w¹¹¹⁸ (m) or aln² (n) flies stained for ATF4 (m, n) or DNA (m’, n’) as quantified in (o). Bar graph shows ATF4 levels in retinas (o) and heads (p) and the number of separate retinas pooled from three independent experiments. Statistical significance was assessed using 2-tailed t test. Bar graphs show (n) and median with interquartile range. ****p < 0.0001. Scale bars in (n’) is 100 µm is same for (m, n). Re: retina, La: lamina. Scale bars in (k) and (n’) are 100 µm and is the same for (h–k) and (m, n). Genotypes are listed in Supplementary File 2. Source data are provided as a Source Data file.