dMtmr6 is essential for development and survival. (A) Schematic representation of CG3530/dMtmr6. Vertical arrows indicate the CRISPR-targeted sites and the resulting deletion. Horizontal arrows indicate P-element insertion site and RNAi target sites. (B) Quantification of the percentage of animals that survive beyond the pupal stage from ubiquitous knockdown experiments (left) or mutant analyses (right). (C) Western blot from whole larvae (feeding L3) and quantifications of the ratio of ref(2)p to actin (C′) and lipidated Atg8a-II to cytosolic Atg8a-I (C″) from the indicated genotypes. (D) Feeding L3 midguts with dMtmr6-null clone cells (white dashed outline, GFP negative) within WT tissue (green) stained with antibody to detect Atg8a and Hoechst to detect nuclei (blue). (E) Quantification of the number of control (FRT42D, +/FRT42D, +) and mutant (FRT42D, dMtmr6Δ/FRT42D, dMtmr6Δ) clone cells generated in midgut (left) and fat body (right) from whole larvae (feeding L3). Data are presented as percentage of n > 100 animals from three experimental replicates (B); mean ± SD, n = 2 animals, three experimental replicates (C); and individual values, n ≥ 3 animals, three experimental replicates (E). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001), using one-way ANOVA (C) and paired, two-tailed t test (E and F). Scale bars, 20 µm. n.s., not significant.