Figure 4. ndi1 expression in the intestine produces alterations in systemic metabolic signaling pathways. (A-B) Western blot (A, Figure S4A) and densitometric analysis (B) of AMPKα phosphorylation at Thr184. AMPKα phosphorylation (normalized to a loading control, beta-Actin)is significantly decreased in flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1), relative to isogenic controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, t test, 5 replicates per condition, 15 flies per replicate). (C) Transcript levels of downstream targets of dFOXO. 4E-BP, InR, l(2)efl, and ImpL2 transcript levels are significantly lower in flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) relative to isogenic controls (“+”, esgAL4>+) at day 10 of adulthood. (**p<0.01, t test, 5 replicates per condition, 5 flies per replicate). (D-E) Western blot (D, Figure S4B) and densitometric analysis (E) of AKT phosphorylation at Ser505 or Thr423, and total AKT levels. AKT phosphorylation (normalized to total AKT) is not altered in flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) relative to isogenic controls (“+”, esgAL4>+) at day 10 of adulthood. Total AKT levels (normalized to beta-Actin) are also unchanged. (n.s., t test, 5 replicates per condition, 5 flies per replicate). (F) Transcript levels of head dilp genes. dilp2, dilp3, and dilp5 transcript levels from heads of flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) are significantly lower than those of controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, **p<0.01, ***p<0.001, t test, 5 replicates per condition, 30 heads per replicate). (G-H) Immunofluorescence staining (G) and quantification (H) of DILP2 levels in insulin producing cells (IPCs). DILP2 fluorescence in IPCs of flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) are significantly lower compared to that of controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, t test, at least 170 IPCs from 12 different brains). (I-J) Transcript levels of sNPF (I) and its cognate receptor, sNPFR1 (J) in heads. Flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) have significantly lower sNPF and sNPFR1 transcript levels in heads than controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, **p<0.01, t test, 4-5 replicates per condition, 30 heads per replicate).