Research Paper Volume 5, Issue 9 pp 662—681

Figure 2. ndi1 expression maintains intestinal homeostasis during aging. (A) Immunofluorescence images evaluating intestinal homeostasis during aging. Control flies (esgGAL4>+, upper panel) and ndi1 expressing flies (esgGAL4>ndi1, lower panel) were assayed for esg⁺ cells (GFP⁺ cells) and mitotic cells (pHH3⁺ cells, arrows) 10 days and 50 days post eclosion. Scale bars=50μm. (B) Quantification of proportion of esg⁺ cells. The proportion of esg⁺ cells (GFP⁺ cells) in all cells (DAPI stain) was increased in aged control flies (“+”, esgGAL4>+), but not in ndi1 expressing flies (“ndi1”, esgGAL4>ndi1). (*p<0.05, ***p<0.001, One-way ANOVA with Tukey's post hoc test, at least 22 flies per condition). (C) Quantification of mitotic cells per field of view. The median number of mitotic events (pHH3⁺ cells per field of view) is elevated in aged control flies (“+”, esgGAL4>+), but not in ndi1 expressing flies (“ndi1”, esgGAL4>ndi1). (*p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test followed by Dunn's multiple comparisons, at least 22 flies per condition). (D)Quantification of ROS levels per area in ISCs/EBs. ndi1 expression (“ndi1”, esgGAL4>ndi1) decreases DHE fluorescence within esg⁺ cells(GFP⁺ cells, arrows in figure S2A) in both 10 and 30 day old intestines compared to isogenic controls (“+”, esgGAL4>+).(*p<0.05, **p<0.01, t test, at least 3 images per gut, 10 guts per condition). (E) Quantification of ROS levels per area in midguts. ndi1 expression in ISCs/EBs (“ndi1”, esgGAL4>ndi1) results in decreased DHE fluorescence in gut tissues relative to isogenic controls (“+”, esgGAL4>+) at 30 days post eclosion. (***p<0.001, t test, at least 3 images per gut, 10 guts per condition). (F) Proportion of flies showing loss of intestinal integrity as a function of age, assayed using blue dye no. 1. Aged flies that express ndi1 in ISCs/EBs (esgGAL4>ndi1) show reduced levels of intestinal barrier dysfunctionrelative to controls (esgGAL4>+). (***p<0.001, binomial test, at least 190 flies per condition). (G) Proportion of flies showing loss of intestinal integrity as a function of age in 5961GS>ndi1 flies with or without RU486-mediated transgene induction. Adult-onset expression of ndi1 by RU486 exposure (0.5mg/l) improves maintenance of intestinal integrity during aging. (H) Systemic expression of Drosomycin, Drosocin and Diptericin in 10 and 45 day old flies. Aged flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) show reduced expression of antimicrobial peptides (AMPs) relative to controls (“+”, esgGAL4>+). (**p<0.01, ***p<0.001, t test, 5 replicates per condition, 5 flies per replicate).