Extended Data Fig. 5: The activities of Tor and Gcn2 signalling pathways modulated by EAA deprivation regulate CNMa expression in the gut, but have no effect on CNMa expression in the brain or fat body.
From: Response of the microbiome–gut–brain axis in Drosophila to amino acid deficit
a, Two-choice behavioural preferences of amino-acid-deprived flies in which UAS-CNMaRNAi had been expressed specifically in the enterocytes (NP1-Gal4), fat body (Cg-Gal4) or neurons (Nsyb-Gal4). Flies carrying tissue-specific GAL4 alone were used as controls. b, c, Western blot analysis of phosphorylated eIF2α (p-eIF2α) and phosphorylated ribosomal protein S6K (p-S6K) in whole-gut lysates collected from mated female flies (around 5–6-days old) that were deprived of EAA (−EAA) for 48 h (b). The intensity of each band corresponding to phosphorylated eIF2α and S6K was quantified using the ImageJ automated digitizing program (NIH) (c). Results from three independent experiments were normalized against those in control flies, the average of which is arbitrarily set as 1. d, Quantifications of GFP fluorescence in the R2 region of the anterior midgut of CNMa-Gal4>UAS-GFP flies (control) or control flies carrying either UAS-mRFP, UAS-ninaBRNAi, UAS-Gcn2RNAi, UAS-Atf4RNAi, UAS-TorWT or UAS-MitfRNAi. e, f, Levels of CNMa expression in the brain or fat body of flies carrying CNMa-Gal4 and UAS-GFP that also harboured either UAS-TorWT or UAS-Gcn2RNAi. GFP signal intensities in the fat body (e) or brain (f) were measured by the native GFP fluorescence. Data are mean ± s.e.m. P values are indicated; unpaired two-tailed t-test in a, c; one-way ANOVA with Tukey’s post-hoc test in d–f. Sample sizes and statistical analyses are shown in Supplementary Table 1.
