nuclear DAPI stain, and panel E a merged picture with all four channels. Also when both stainings are performed separately on different batches of tissue from larvae of the indicated genotype, the nuclear 3xHA-FKH is detected by aFKH1 as well as anti-HA (data not shown). This demonstrates that aFKH1 is a suitable tool to visualize FKH in immunostainings, and that the main nuclear signal detected by the antibody corresponds to FKH protein and not an unspecific protein recognized by the antibody. In contrast to the endogenous protein (see figure 4), overexpressed FKH is localized in the nucleus also under conditions of high TOR and insulin signaling. The fatbody shown in panels B–E is from a population of larvae which had been reared on protein-rich yeast paste, nevertheless the overexpressed 3xHA-FKH protein is nuclear. We also investigated the subcellular localization of overexpressed fluorescent FKH fusion proteins in cultured cells. (F–H) In S2R+ cells that had been transiently co-transfected with pAct5C-GAL4 and pTRW-FKH and were growing in serum-containing medium, the red fluorescent mRFP1-FKH protein is localized in the cell nuclei. (I–K) Likewise, in S2R+ cells that had been transiently transfected with pAGW-FKH, were growing in serum-containing medium and had been furthermore stimulated with 100 nM bovine insulin for 20 min., the green fluorescent EGFP-FKH is nuclear. Panels F and I show the signal of the respective fluorescent FKH fusion proteins, panels G and J the DAPI DNA stainings of the same confocal sections, and H and K the merged pictures containing signals from both channels. The same nuclear localization was observed in cells that had either been treated with 20 nM rapamycin for 30 min. or serum-deprived over night and subsequently subjected to PI3K inhibition with 50 mM LY294002 for 1 h (data not shown). (TIF) Figure S3 Induction of CG6770 promoter activity by FKH. Over-expressed FKH protein activates transcription from the CG6770 promoter in cultured cells. S2R+ cells were transiently tranfected with a reporter plasmid containing the firefly luciferase gene under control of the CG6770 regulatory region. The Renilla luciferase construct polIII-RL was co-transfected as an internal control to compensate for well-to-well variation in transfection efficiency. Before lysis and luciferase measurements, cells were incubated in serum-free medium over night to lower growth factor signaling levels. Compared to cells transfected with the luciferase vectors only, co-transfection of the dFOXO expression plasmid pAHW-dFOXO-Blast lead to a several fold induction of luciferase expression from the CG6770 promoter. Expression of FKH by co- transfection with pAHW-FKH-Blast elicited a much stronger induction of the reporter construct, leading to luciferase levels that were 5 fold higher than in the dFOXO-expressing cells and 20 fold higher compared to the control cells without expression vector. (TIF) References 1. Carlsson P, Mahlapuu M (2002) Forkhead transcription factors: key players in development and metabolism. Dev Biol 250: 1–23. 2. Hannenhalli S, Kaestner KH (2009) The evolution of Fox genes and their role in development and disease. Nat Rev Genet 10: 233–240. 3. Van Der Heide LP, Hoekman MF, Smidt MP (2004) The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem J 380: 297–309. 4. Lin K, Dorman JB, Rodan A, Kenyon C (1997) daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319–1322. 5. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, et al. (1997) The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389: 994–999. 6. Friedman JR, Kaestner KH (2006) The Foxa family of transcription factors in development and metabolism. Cell Mol Life Sci 63: 2317–2328. Regulation of Drosophila FKH/FOXA by TOR Figure S4 Correlation with TSC1/2 and Rheb gain-of-function phenotypes. Inhibition or activation of TOR signaling leads to similar phenotypes as FKH overexpression and knockdown, respectively. (A and C) On a protein-rich yeast paste diet, co- expression of TSC1 and TSC2 in cell clones in the larval fatbody leads to a strong reduction in cell size. (B) The growth-inhibiting effect of TSC1/2 expression is much less pronounced in starved animals. (D) Conversely, activation of TOR signaling by expression of the small GTPase Rheb (Saucedo et al., 2003; Stocker et al., 2003) has a very mild growth-promoting effect on a protein-rich diet and (E and F) a stronger one under conditions of starvation. The same driver line was used as in the experiments shown in figure 2. A similar correlation was observed when using the expression of FKH target gene candidates as a readout. (G) Like FKH knockdown, expression of Rheb (driven by arm-Gal4) silences transcription of CG6770 and d4E-BP. Inhibition of TOR signaling by TSC1/2 expression (driven by ppl-Gal4) leads to a strong elevation of mRNA levels of both genes, as does FKH expression. These observations further strengthen our model that FKH is functionally linked to the TOR signaling module. (TIF) Sequences of primers used in this study. Text S1 (PDF) Text S2 Regions in the fkh mRNA sequence targeted by the individual RNAi constructs used. (PDF) Text S3 Location of the peptide used for antibody generation within the FKH protein sequence. (PDF) Acknowledgments We thank Thomas Neufeld (University of Minnesota), Ernst Hafen and Hugo Stocker (ETH Zu¨rich), the Bloomington Drosophila Stock Center and the TRiP (Transgenic RNAi Project) at Harvard Medical School (NIH/ NIGMS R01-GM084947) for providing fly stocks, Norbert Perrimon (Harvard Medical School), Barry Dickson (IMP Vienna) and the Drosophila Genomics Resource Center (Indiana University) for plasmids used in this study. Author Contributions Conceived and designed the experiments: MHB RA MJP MAJ. Performed the experiments: MHB. Analyzed the data: MHB MAJ. Contributed reagents/materials/analysis tools: MAJ. Wrote the paper: MHB MAJ. Generated DNA constructs: MAJ. 7. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, et al. (2003) Influence of TOR kinase on lifespan in C. elegans. Nature 426: 620. 8. Hansen M, Taubert S, Crawford D, Libina N, Lee SJ, et al. (2007) Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6: 95–110. 9. Meissner B, Boll M, Daniel H, Baumeister R (2004) Deletion of the intestinal peptide transporter affects insulin and TOR signaling in Caenorhabditis elegans. J Biol Chem 279: 36739–36745. 10. Panowski SH, Wolff S, Aguilaniu H, Durieux J, Dillin A (2007) PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature 447: 550–555. 11. Sheaffer KL, Updike DL, Mango SE (2008) The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging. Curr Biol 18: 1355–1364. 12. Lee HH, Frasch M (2004) Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns. Dev Dyn 229: 357–366. PLoS ONE | www.plosone.org 12 December 2010 | Volume 5 | Issue 12 | e15171