(A) Schematic of the experimental setup to generate temporal knockdown of foxo in wild-type or Tsc1 mutant clones, and isolation of single clones using LCM at 108 hr after egg laying (AEL) from normal food and 156 hr AEL from NR. Solid or dashed lines represent clones isolated from larvae shifted to 30°C for 12 hr or maintained at 25°C, respectively. (B) FoxO staining of eye imaginal discs with Tsc1 mutant clones dissected from larvae raised on normal food at 25°C, 30°C or shifted from 25°C to 30°C for 12 hr. Clones are negatively marked by GFP, and DAPI stains nuclei. Scale bar = 50 µm. (B’) Quantification of ratio of nuclear FoxO intensity in Tsc1 mutant clone over wild-type from larvae raised on normal food or NR at temperatures described in B. n > 9. Data are represented as mean ± SD. **p<0.01, ***p<0.001 and ns = not significant. (C) Venn diagram depicting number of genes, upregulated and downregulated, between all conditions tested. p<0.0025 and FDR < 0.2. (C’) Gene ontology analysis of the downregulated genes in Tsc1 mutant cells upon foxo knockdown as compared to Tsc1 mutant cells under NR.