Fig. 3: Autophagy sets a limit to Notch accumulation and Notch-dependent crystal cell differentiation.

Crystal cells were visualized in Z-stack projections of lymph gland single lobes (dashed lines) by anti-PPO immunofluorescence (A–H); RNAi expression was driven with domeMESO-Gal4. I–M upper row: Anti-Notch immunofluorescence in wild type larvae and lymph gland lobes with reduced autophagy. N Quantification of the normalized number of crystal cells per lymph gland lobe. Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. Mean values of genotypes marked with different letters are significantly different (p < 0.05). For wild type, n = 33 primary lobes; for Atg1^RNAi, n = 43; for N^RNAi, n = 18; for N^RNAi/Atg1^RNAi, n = 23; for N^55e11/+, n = 14; for N^55e11/+; Atg1^RNAi, n = 28, for Su(H)¹/+, n = 18; for Su(H)¹/+; Atg1^RNAi, n = 18. I–M lower row: Notch transcriptional activity was evaluated with an E(spl)mβ-HLH-GFP transcriptional reporter in each genotype. O Mean fluorescence intensity of anti-Notch staining was quantified in single slices, and relative change of immunofluorescence respect to the control was calculated and plotted. Statistical analysis in was performed using one-way ANOVA followed by Dunnett’s test for comparisons of treatments versus control (p < 0.0001 for each genotype). For wild type, n = 37 primary lobes; for Atg1^RNAi, n = 25; for Atg17^RNAi, n = 16; for Vps15^RNAi, n = 16; for Atg18^RNAi, n = 25. P Quantification of the normalized number of GFP-positive cells per lobe. For wild type, n = 42 primary lobes; for Atg1^RNAi, n = 21; for Atg17^RNAi, n = 20; for Vps15^RNAi, n = 55; for Atg18^RNAi, n = 13. Statistical analysis in was performed using Kruskal-Wallis test followed by Dunn’s test for comparisons of treatments versus control (p = 0.0036; p = 0.0004; p < 0.0001; p < 0.0001, respectively). N–P Box plots show the data distribution, with the box representing the interquartile range (25^th and 75^th percentiles), the central line indicating the median, and whiskers extending to the minimum and maximum values. Source data for plots are provided as a Source Data file. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.