Figure 6. Loss of Atg9 enhances TOR activity in Drosophila adult midgut.

(A) The adult midguts of denoted genotypes were dissected, lysed, and subjected to Western blot analysis with anti-Atg9, anti-Ref(2)P, anti-p-S6K, anti-p-4EBP and anti-tubulin antibodies. (B) Inhibition of TOR activity by feeding flies with rapamycin rescued Atg9 mutant midgut defects. (C) Quantification of posterior midgut cell size shown in (B). n ≥ 17, data are mean ±s.e.m. ***p<0.001. (D–K) Atg9 genetically interacts with components of the TOR signaling pathway. The Atg9^RNAi-induced midgut defects (D) could be suppressed by the coexpression of TSC1-TSC2 (E), Rheb^RNAi (F), dominant-negative TOR (TOR^TED) (G), or dominant-negative S6K (S6K^KQ) (H), whereas coexpression of TOR activator Rheb (I) or knock-down of TSC1 (J) or TSC2 (K) could not rescue the Atg9^RNAi-induced midgut defects. Genetic analyses were performed for three times with 100% penetrance of the phenotype. (L) Quantification of posterior midgut cell size shown in (D–K). n ≥ 18, data are mean ±s.e.m. ***p<0.001. ns, not statistically significant. Scale bar: 20 μm.

Figure 6—figure supplement 1. Rapamycin treatment rescues the intestinal barrier dysfunction of Atg9 mutants.

(A) Quantification of Smurf flies showing that rapamycin treatment significantly decreased the number of Smurf-positive flies in aged Atg9 mutants. n ≥ 120, data are mean ±s.e.m., **p<0.01. ns, not statistically significant. (B) Inhibition of TOR activity by feeding flies with rapamycin could not rescue the short lifespan of Atg9 mutants, compared with controls. (Long-rank test, p<0.001).

Figure 6—figure supplement 2. Atg9 genetically interacts with components of the insulin receptor/phosphoinositide 3-kinase (InR/PI3K) signaling pathway.

The Atg9^RNAi-induced midgut defects (A) could be suppressed by the coexpression of InR^RNAi (B), PI3K^RNAi (C), or Akt^RNAi (D), whereas coexpression of PI3K (E) or Akt (F) dramatically enhanced Atg9^RNAi-induced midgut defect. Genetic analyses were performed for three times with 100% penetrance of the phenotype. The cell size of posterior midgut ECs of each genotype was quantified. n ≥ 16, data are mean ±s.e.m. ***p<0.001, *p<0.05. ns, not statistically significant. Scale bar: 20 μm.