PKB-mediated inhibition of FoxO is lost in Tsc1 mutant cells under NR.

(A) Phospho-PKB staining (in red) of eyes discs with hsFlp Tsc1 and PTEN (A’) mutant clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR. Scale bars are 75 μm. (B) Western blot analysis of proteins extracted from the control or Tsc1 mutant eye imaginal discs. The extremely small size of the control eye discs from animals reared on NR precluded the Western analysis. Total levels of PKB were unaltered. β-tubulin served as loading control. (C) FoxO staining (in magenta) of eyes discs with hsFlp control, Tsc1 and PTEN clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR. Scale bars are 75 μm. (D) Eyes bearing control, Tsc1 knockdown or PTEN knockdown tissue, with or without the overexpression of FoxO, of animals reared on normal food or NR. Scale bars are 100 μm.