Fig. 5: Knock-down of elements of ESCRT complexes resulted in increased Notch abundance and enhanced crystal cell differentiation.

Anti-Lozenge (Lz) and anti-prophenoloxidase (PPO) stainings to visualize crystal cells in Z-stack projections of lymph gland primary lobes (white dashed lines), and assess the effect of domeMESO-Gal4-driven knock-down of genes of different ESCRT complexes (B–D) or the gene Vps4 (E) that encodes the effector ATPase of ESCRT complexes, in comparison with wild type individuals (A). F, G Quantification of normalized crystal cells per lobe as assessed by anti-Lz or anti-PPO staining respectively. F One-way ANOVA followed by Dunnett’s test for treatment versus control comparisons (p < 0.0001; p = 0.0125; p < 0.0001; p < 0.0001, respectively). G Two-sided likelihood ratio test (Chi-squared) followed by Dunnett’s test for treatment versus control comparisons (p < 0.0001 for each genotype). For wild type, n = 86 primary lobes; for TSG101^RNAi, n = 24 for Lz and n = 22 for PPO; for Vps25^RNAi, n = 24; for shrub^RNAi, n = 19; for Vps4^RNAi, n = 33. Notch immunofluorescence performed with an antibody targeting the Notch extracellular domain shows that Notch protein levels in each genotype (A–E) parallels the increase in differentiation of crystal cells. H Quantification of Notch immunofluorescence expressed as relative change in fluorescence intensity respect of the wild type genotype. For wild type, n = 20; for TSG101^RNAi, n = 16; for Vps25^RNAi, n = 19; for shrub^RNAi, n = 15; for Vps4^RNAi, n = 14. One-way ANOVA followed by Dunnett’s test for treatment versus control comparisons (p < 0.0001; p = 0.0038; p < 0.0001; p = 0.0732, respectively). F–H Box plots show the data distribution, with the box representing the interquartile range, 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 < 0.0001; ns, not significant (p > 0.05).