42 g fresh yeast in 6.5 ml PBS). The fatbody samples shown in Figure 2 are derived from larvae that were treated as follows: for the ‘‘yeast’’ condition, larvae were kept on yeast paste until 64 h AED. For the ‘‘PBS’’ condition, larvae were kept on yeast paste until 64 h AED, washed out of the yeast paste, rinsed with water and transferred to a petri dish with PBS-soaked filter paper for another 24 h. For the ‘‘rapamycin’’ condition, larvae were kept on yeast paste until 48 h AED, before 200 ml of a 50 mM rapamycin (LC Laboratories) solution was added to the yeast paste and the plates were further incubated for 24 h at 25uC. Immunohistochemistry Antibody stainings were performed as described before [43]. In brief, larval tissues were dissected in Drosophila Ringer’s solution and fixed for 309 in 4% formaldehyde in PBS+0.5% Tween-20 (PBT). Tissue was blocked with 5% Goat Serum in PBT for 309, and washed before and after incubation with primary antibody in PBS+0,5% Tween-20 or PBS+0.1% Tween-20, respectively, for 5/5/15/309. Incubation with primary antibodies diluted in blocking solution was performed over night at 4uC. Fluores- cence-coupled secondary antibodies were applied for 1 h at RT. DAPI (1 mg/ml) was included in the last washing step before samples were mounted in Mowiol (Roth). Primary antibodies used were rabbit aFKH1 (dilution 1:200), mouse anti-GFP (Sigma, dilution 1:500), guinea pig anti-ppl (a gift of Ingo Zinke, dilution 1:500) and mouse anti-CD2 (Serotec, dilution 1:200). Secondary antibodies (all diluted 1:200 in PBS+0.1% Tween-20+5% Goat Serum) used were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) and Cy5 anti-guinea pig (Jackson ImmunoResearch). Mounted tissue was analyzed using a Zeiss LSM 710 confocal microscope and images were further processed with the Zeiss LSM Image Software. The size of cell clones in fatbody samples was measured using the ImageJ software. Cell culture and luciferase assays Drosophila S2R+ cells were obtained from the Drosophila Genomics Resource Center (DGRC stock nr. 150), cultured at 25uC in Schneider’s Drosophila medium (Invitrogen) containing 10% heat-inactivated FCS (Sigma). Cells were split and diluted to a density of 16106 per ml once per week. For reporter gene assays, S2R+ cells were transfected with the FuGene HD reagent (Roche) in 24 well-plates (Greiner). Per well, 50 ng of the reporter construct pGL3-CG6770 and 5 ng polIII-RL were used. Where indicated, 200 ng of expression plasmids pAHW-FKH-Blast or pAHW-dFOXO-Blast were co-transfected with the reporter gene constructs. After the cells had grown in the multiwell plates to a density of ca. 80%, transfection was performed for 8 h, after which cells were allowed to recover on serum-containing medium for 12 h and subsequently subjected to serum deprivation over night. Cells were lysed in Passive Lysis Buffer before measuring firefly and Renilla luciferase activities subsequently in each sample according to the Dual Luciferase System protocol (Promega) in a Wallac luminometer (PerkinElmer). The firefly luciferase values were normalized to the Renilla values to account for variations in transient transfection efficiency. Realtime PCR Quantitative realtime PCR to measure transcript levels was done as described previously [20]. Briefly, total RNA was extracted from Drosophila larvae with TriFast (peqlab)/chloroform in a Precellys homogenisator (peqlab), following the manufactur- er’s protocol. Extracted RNA was dissolved in DEPC-treated water. To minimize RNA degradation, RNA was stored at 280uC Regulation of Drosophila FKH/FOXA by TOR and used as a template for cDNA synthesis within 24 h. 1 mg of total RNA was used for cDNA synthesis with the QuantiTect kit (Qiagen). cDNA integrity and absence of contamination by genomic DNA was assessed by a PCR amplification of Actin5C from + and 2 reverse transcriptase reactions. Realtime qPCR was performed with a CFX96 instrument (Bio-Rad). Primers were designed with the GC-content recommended for qPCR (between 40% and 60%) and low self-complementarity using the software tool Primer3. Prior to quantitation experiments, primer efficiency and NTC (No Template Control) was tested for each primer pair to ensure an efficiency of at least 80% and rule out primer dimer formation. PCR reactions consisted of first-strand cDNA template and iQ SYBR Green Supermix (Biorad). Actin5C and rp49 transcript levels were used for normalization. All primer sequences are provided as supporting information in Text S1. For every mRNA quantitation, the minimal number of replicates consisted of biological triplicates, with each triplicate measured twice as technical replicates for a total of six measurements. Statistics Error bars represent the standard error of the mean (SEM) if not stated otherwise. Statistical significance was assessed using an unpaired two-tailed Student’s t-test, comparing the experimental data with the respective controls. For comparisons between one experimental value and two control values (such as wildtype and unspecific RNAi controls), analysis of variance (ANOVA) was used in addition to confirm the significance values derived from the pairwise t-tests. If not stated otherwise, all experiments were carried out at least twice independently. Asterisks indicate a p- value of ,0.05 (*), ,0.01 (**) or ,0.001 (***). Significance was tested with the software InStat 3 from GraphPad Software. Supporting Information Figure S1 Efficiency of RNAi-mediated knockdown. fkh mRNA levels were quantified in larval extracts by quantitative realtime PCR to control RNAi-mediated knockdown. Compared to wild- type larvae, expression of fkh dsRNA (pMF3-fkh construct) under control of the armadillo driver lead to a reduction of fkh transcript levels by 60%. Heterozygosity for the fkh1 allele reduced larval transcript levels by 70% compared to wildtype animals. (TIF) Figure S2 Control of antibody specificity and localization of overexpressed FKH. FKH is the main protein recognized by the newly generated aFKH1 antibody in western blots and immuno- stainings, and overexpressed FKH is constitutively nuclear. (A) Western blot analysis of larval extracts probed with the aFKH1 antibody. Lane 1: in an extract from wild-type larvae, the antibody detects a single protein band of approximately 54 kDa, which is the predicted molecular weight of FKH and therefore most likely corresponds to endogenous FKH. Lane 2: in an extract from w; UAS-mRFP1-FKH; ppl-GAL4 larvae, the 54 kDa protein is detected as well. In addition, a band of higher molecular weight is visible which corresponds to the transgenically encoded mRFP1-FKH fusion protein. (B–E) Immunofluorescent double staining of fatbody from y w hs-FLP;; Act.CD2.Gal4 UAS-GFPnls/UAS-3xHA-FKH larvae. The GFP-marked cell clones express transgenically encoded 3xHA-tagged FKH protein, which is recognized by a mouse monoclonal anti-HA antibody (B) as well as the rabbit polyclonal aFKH1 antibody (D). Secondary antibodies used were anti-mouse- Cy3 and anti-rabbit-Cy5. For both antibodies, one panel shows the only the signal of the actual immunostaining (B and D), panel C shows the merged signals of the clone marker GFP and and the PLoS ONE | www.plosone.org 11 December 2010 | Volume 5 | Issue 12 | e15171