dMtmr6 regulates autophagic flux independently of Atg1. (A) Representative Western blot from fat body of indicated genotypes to measure levels of phosphorylated p70-S6K (A′) and phosphorylated AKT (Av) proteins relative to actin. (B) Representative Western blot using fed L3 fat body from fat body–specific knockdown of the indicated genotypes (CG-Gal4 > UAS-RNAi) to measure the ratio of lipidated to cytosolic Atg8a (B′) and ref(2)p to actin (B″) protein levels. (C, F, and G) Representative images of starved L3 fat body with control (GFP-negative) and knockdown cells of indicated genotypes (white dashed outline, green cells) stained with an antibody to detect Atg8a and Hoechst to detect nuclei (blue). Atg8a only is depicted in C′, F′, and G′, and red boxes indicate areas shown in zoom panels at right. (D and E) Quantification of cellular Atg8a puncta number (D) and average size (E) as represented in C, F, and G. Data are presented as mean ± SD, n = 6, three experimental replicates (A and B) or mean ± minimum to maximum (n ≥ 9; D and E). Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001), using unpaired two-tailed t test (A) and one-way ANOVA (B, D, and E). Scale bars, 20 µm.