Drosophila stocks and culture
The following strains were obtained from the Bloomington Drosophila Stock Center: OreR, w1118
, UAS-p35, and tub-Gal80ts
was obtained from the Vienna Drosophila RNAi Center (transformant ID 106097). NP1Gal4 was kindly provided by D. Ferrandon; UAS-Magro by C.Thummel; the foxoW24
mutant allele by M.Tatar and the foxoΔ94
allele by L. Partridge; GMRGal4, UAS-HepACT
by M. Mlodzik; esg-Gal4 and esg-LacZC4-1
by S. Hayashi; Su(H)-GBE-lacZ by S. Bray; puc-lacZ
) by E. Martín-Blanco. UAS-Foxo was previously described in (Puig et al., 2003
). For aging and starvation sensitivity experiments, UAS-FoxoRNAi
, UAS-Magro, and UAS-Foxo transgenenic lines were backcrossed 8-10 generations into the w1118
background. Backcrossed UAS flies and w1118
male siblings were used to set up the crosses in order to reduce genetic background effects. The foxoW24
mutant was backcrossed into the yw
background, and yw
siblings were used as controls ().
All flies were raised on standard yeast and molasses - based food, at 25°C and 65% humidity, on a 12 h light/dark cycle, unless otherwise indicated. Food for high-sugar/low-yeast (HSLY) diet feeding (and standard sugar/yeast (St. SY) control food) was made with the following protocol (as described in (Skorupa et al., 2008
)): HSLY – 1.5 g agar / 40 g sucrose / 2.5 g yeast (yeast flake) / 0.3 mL propionic acid / 100 mL water; St. SY – 1.5 g agar / 10 g sucrose / 10 g yeast / 0.3 mL propionic acid / 100 mL water. Ingredients were combined, heated to at least 120°C, and cooled before pouring.
Analysis of gene expression
Total RNA from 8-10 guts or from 5 whole flies (without heads) was extracted using Trizol and cDNA synthesized using Superscript II (Invitrogen). Real time PCR was performed using SyberGreen, a Biorad IQ5 apparatus and the primers pairs described in Supplemental Information
). Results are average +/− standard error of at least 3 independent samples, and quantification of gene expression levels were measured relative to the expression of Actin5c.
Western Blot analysis
Guts (10-20) were homogenized in protein sample buffer; proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane using standard procedures. The following antibodies (all from Cell Signaling) were used: phsopho-Akt (anti-p-Drosophila Akt (S505); rabbit, 1:1000), total Akt (anti-pan Akt; rabbit, 1:1000), and β-actin (anti-beta-actin; rabbit, 1:5000). Signal was detected using HRP-conjugated anti-rabbit and chemi-luminescence (Pierce), according to manufacturer instructions.
Detection of β-galactosidase activity
Intact guts were dissected in PBS + 2mM MgCl2 and fixed for 10 minutes in 0.5% glutaraldehyde. Detection of β-galactosidase activity was carried out at room temperature in staining buffer (PBS, 2 mM MgCl2, 5 mM K4(Fe[CN]6), 5 mM K3(Fe[CN]6), 0.1% X-gal).
For TAG assays, 4 to 5 females (without the head) were homogenized in 150μl of PBST (PBS, 0.1% Tween 20), heated at 70°C to inactivate endogenous enzymes (homogenate cleared by centrifugation). 30μl of cleared extract was used to measure triglycerides concentrations according to the manufacturer instructions (StanBio Liquicolor Triglycerides). TAG levels were normalized to weight (measured using MT XS64 scale).
Oil Red O staining
Guts were dissected in PBS and fixed in 4% formaldehyde/PBS for 20 minutes. Guts were then washed twice with PBS, and incubated for 30 minutes in fresh Oil Red O solution (6 mL of 0.1% Oil Red O in isopropanol and 4 mL DEPC water; and passed through a 0.45 –μm syringe), followed by rinsing with distilled water.
Immunostaining and Microscopy
Intact guts were fixed at room temperature for 45 minutes in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM Sodium Phosphate, 1 mM MgCl2, 4% formaldehyde. All subsequent incubations were done in PBS, 0.5% BSA, 0.1% TritonX−100 at 4°C.
The following primary antibodies were used: mouse anti-β-Gal (1:200), mouse anti-Prospero (1:250), and mouse anti-Armadillo (1:100) from Developmental Studies Hybridoma Bank. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. Hoechst was used to stain DNA.
Confocal images were collected using a Leica SP5 confocal system and processed using the Leica software and Adobe Photoshop.
Ex vivo insulin stimulation
Guts (8-10) were dissected in PBS, and then transferred to M3+BPYE (without serum) cell culture media with or without bovine insulin (0.1 units per 40 μL media, from Sigma) for 20 minutes. Samples were then prepared for protein extraction / Western blot analysis as previously described.