Cloning of LexO-PtenRi
The sequence targeted by LexO-PtenRi was PCR-amplified from y w genomic DNA using primer set PCR1, as used for the UAS-PtenRi construct by VDRC. Restriction digestion sites for EcoRI and XbaI were introduced by using primer set PCR2, and IR was generated by digesting with EcoRI and self-ligation. The IR construct was subcloned into pLOTattB by using the XbaI site to give pLOTattBLexO-PtenRi.
All crosses were maintained at 25°C on normal fly food unless otherwise stated. Normal fly food is composed of 100 g fresh yeast, 55 g cornmeal, 10 g wheat flour, 75 g sugar, 8 g bacto agar, and 1.5% antimicrobial agents (33 g/L nipagin and 66 g/L nipasol in ethanol) in 1 L water. NR food was prepared by reducing the amount of yeast to 10% with 1.5% nipagin (100 g/L in ethanol).
Fly lines used: LexO-PtenRi line was generated in this study by injecting the pLOTattBLexO-PtenRi plasmid into line ΦX-22A; dpp>LHG, Act>CD2>LHV2 and LexO-mCherry-CAAX UAS-CD8-GFP tub-Gal80ts; coinFLP-LexGAD/Gal4 (59270 Bloomington Drosophila Stock Center (BDSC)); foxoRi (107786 VDRC); Tsc1Ri (31039 BDSC).
Immunofluorescence and microscopy
Adult wings of females were detached from thorax using forceps 4 days after eclosion and mounted in Euparal (Roth #7356.1). They were imaged on KEYENCE VHX1000 digital microscope. Imaginal discs were dissected 108 h AEL from normal food and 156 h AEL from NR unless otherwise stated.
Imaginal discs and brains were fixed in 4% paraformaldehyde (PFA, 30 min, room temperature (RT)), washed thrice in 0.3% Triton-X in PBS (PBT, 15 min, RT), blocked in 2% Normal Donkey Serum in 0.3% PBT (2 h, 4°C), incubated with primary antibodies (overnight, 4°C), washed thrice in 0.3% PBT (15 min each, RT), incubated with secondary antibodies (2 h, RT), washed thrice in 0.3% PBT (15 min each, RT), stained with DAPI in 0.3% PBT (1:2000, 10 min, RT), and washed once with PBS (10 min, RT). The samples were mounted in VECTASHIELD (Vector Laboratories H-1000). Confocal images were obtained on Leica SPE TCS confocal laser-scanning microscope.
Antibodies used: rabbit anti-phospho-Drosophila Akt Ser505 (Cell Signaling Technology #4054, 1:300), rabbit anti-Repo (gift from Angela Giangrande, 1:500), rabbit anti-cleaved Drosophila Dcp-1 Asp216 (Cell Signaling Technology #9578, 1:200), goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific #A11034, 1:500) and goat anti-rabbit Alexa Fluor 633 (Thermo Fisher Scientific #A21070, 1:500). All antibody dilutions were made in 2% NDS in 0.3% PBT.
Quantification and statistical analysis
The area between veins L3-L4 and area of crossveins from adult wings of females were measured using ImageJ software. The area of Gal4 clones was calculated by dividing the GFP-positive area by DAPI area for individual discs using ImageJ software. Error bars in bar plots represent mean ± standard deviation. The student’s t-test (two-tailed) was used to test for significance; *** indicates a p-value <0.001. n for experiments is indicated in corresponding figure legends.