were sequenced and compared by using ClustalW2 software [45] (http://www.ebi.ac.uk/Tools/clustalw2/) and DnaSP 4.0 [46]. The statistical power for the comparison was calculated by GPower software 3.0.3 [47]. Supporting Information Figure S1 Correlation between Microarray and qRT-PCR Results. Found at: doi:10.1371/journal.pgen.1000221.s001 (0.83 MB TIF) Table S1 List of Significantly Altered Genes in Hypoxia- Selected Drosophila melanogaster at Larval Stage. Found at: doi:10.1371/journal.pgen.1000221.s002 (0.23 MB PDF) Table S2 List of Significantly Altered Genes in Hypoxia- Selected Drosophila melanogaster at Adult Stage. Found at: doi:10.1371/journal.pgen.1000221.s003 (0.08 MB PDF) Table S3 List of Significantly Altered Biological Processes in Hypoxia-Selected Drosophila melanogaster at Larval Stage. Found at: doi:10.1371/journal.pgen.1000221.s004 (0.18 MB PDF) Table S4 List of Significantly Altered Biological Processes in Hypoxia-Selected Drosophila melanogaster at Adult Stage. References 1. Jiang C, Xia Y, Haddad GG (1992) Role of ATP-sensitive K+ channels during anoxia: major differences between rat (newborn and adult) and turtle neurons. J Physiol 448: 599–612. 2. Doll CJ, Hochachka PW, Reiner PB (1991) Effects of anoxia and metabolic arrest on turtle and rat cortical neurons. Am J Physiol 260: R747–755. 3. Simon LM, Robin ED (1970) Changes in heart and skeletal muscle cytochrome oxidase activity during anaerobiosis in the freshwater turtle Pseudemys scripta elegans. Comp Biochem Physiol 37: 437–443. 4. Johnston IA (1975) Anaerobic metabolism in the carp (Carassius carassius L.). Comp Biochem Physiol B 51: 235–241. 5. Hochachka PW, Guppy M (1987) Animal anaerobioses. Cambridge, MA: Harvard University Press. pp 10–35. 6. Haddad GG, Sun Y, Wyman RJ, Xu T (1997) Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster. Proc Natl Acad Sci U S A 94: 10809–10812. 7. Haddad GG, Wyman RJ, Mohsenin A, Sun Y, Krishnan SN (1997) Behavioral and Electrophysiologic Responses of Drosophila melanogaster to Prolonged Periods of Anoxia. J Insect Physiol 43: 203–210. 8. Haddad GG, Donnelly DF (1990) O2 deprivation induces a major depolariza- tion in brain stem neurons in the adult but not in the neonatal rat. J Physiol 429: 411–428. 9. Haddad GG, Gandhi MR, Mellins RB (1982) Maturation of ventilatory response to hypoxia in puppies during sleep. J Appl Physiol 52: 309–314. 10. Bier E (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 6: 9–23. 11. Bilen J, Bonini NM (2005) Drosophila as a model for human neurodegenerative disease. Annu Rev Genet 39: 153–171. 12. Haddad GG, Ma E (2001) Neuronal tolerance to O2 deprivation in drosophila: novel approaches using genetic models. Neuroscientist 7: 538–550. 13. Hafen E (2004) Cancer, type 2 diabetes, and ageing: news from flies and worms. Swiss Med Wkly 134: 711–719. 14. Jacobs HT, Fernandez-Ayala DJ, Manjiry S, Kemppainen E, Toivonen JM, et al. (2004) Mitochondrial disease in flies. Biochim Biophys Acta 1659: 190–196. 15. Toma DP, White KP, Hirsch J, Greenspan RJ (2002) Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nat Genet 31: 349–353. 16. White KP, Rifkin SA, Hurban P, Hogness DS (1999) Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179–2184. 17. Zhou D, Xue J, Chen J, Morcillo P, Lambert JD, et al. (2007) Experimental selection for Drosophila survival in extremely low O2 environment. PLoS ONE 2: e490. 18. Li TR, White KP (2003) Tissue-specific gene expression and ecdysone-regulated genomic networks in Drosophila. Dev Cell 5: 59–72. 19. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116–5121. 20. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, Conklin BR (2002) GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat Genet 31: 19–20. Adaptation to Long-Term Hypoxia in Drosophila Found at: doi:10.1371/journal.pgen.1000221.s005 (0.11 MB PDF) Table S5 Primers for qRT-PCR and ChIP-PCR Assays. Found at: doi:10.1371/journal.pgen.1000221.s006 (0.02 MB PDF) Table S6 Summary of Genomic Locations of hairy Transcription Repressor Binding Elements in the cis-Regulatory Regions of Genes Encoding TCA Cycle Enzymes. Found at: doi:10.1371/journal.pgen.1000221.s007 (0.03 MB PDF) Table S7 Comparison of the TPI Genomic Locus between NF and AF with Multiple Sequence Alignment. Found at: doi:10.1371/journal.pgen.1000221.s008 (0.06 MB PDF) Acknowledgments We thank Orit Gavrialov, Juan Wang, Jenna Lau, Nuny Morgan and Ying Lu-Bo for technical assistance. Author Contributions Conceived and designed the experiments: DZ KPW GGH. Performed the experiments: DZ JCKL. Analyzed the data: DZ JX JCKL NJS. Contributed reagents/materials/analysis tools: NJS KPW. Wrote the paper: DZ JX GGH. 21. Doniger SW, Salomonis N, Dahlquist KD, Vranizan K, Lawlor SC, et al. (2003) MAPPFinder: using Gene Ontology and GenMAPP to create a global gene- expression profile from microarray data. Genome Biol 4: R7. 22. Reiling JH, Sabatini DM (2006) Stress and mTORture signaling. Oncogene 25: 6373–6383. 23. Phillips RJ, Mestas J, Gharaee-Kermani M, Burdick MD, Sica A, et al. (2005) Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1alpha. J Biol Chem 280: 22473–22481. 24. Ock J, Jeong J, Choi WS, Lee WH, Kim SH, et al. (2007) Regulation of Toll-like receptor 4 expression and its signaling by hypoxia in cultured microglia. J Neurosci Res 85: 1989–1995. 25. Mehrhof FB, Muller FU, Bergmann MW, Li P, Wang Y, et al. (2001) In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen- activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein. Circulation 104: 2088–2094. 26. Kuhlicke J, Frick JS, Morote-Garcia JC, Rosenberger P, Eltzschig HK (2007) Hypoxia inducible factor (HIF)-1 coordinates induction of Toll-like receptors TLR2 and TLR6 during hypoxia. PLoS ONE 2: e1364. 27. Gunaratnam L, Morley M, Franovic A, de Paulsen N, Mekhail K, et al. (2003) Hypoxia inducible factor activates the transforming growth factor-alpha/ epidermal growth factor receptor growth stimulatory pathway in VHL(2/2) renal cell carcinoma cells. J Biol Chem 278: 44966–44974. 28. Arsham AM, Howell JJ, Simon MC (2003) A novel hypoxia-inducible factor- independent hypoxic response regulating mammalian target of rapamycin and its targets. J Biol Chem 278: 29655–29660. 29. Fan C, Iacobas DA, Zhou D, Chen Q, Lai JK, et al. (2005) Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia. Physiol Genomics 22: 292–307. 30. Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, et al. (2005) Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell 9: 617–628. 31. Jogi A, Ora I, Nilsson H, Lindeheim A, Makino Y, et al. (2002) Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci U S A 99: 7021–7026. 32. Hochachka PW (1986) Defense strategies against hypoxia and hypothermia. Science 231: 234–241. 33. Hochachka PW, Clark CM, Brown WD, Stanley C, Stone CK, et al. (1994) The brain at high altitude: hypometabolism as a defense against chronic hypoxia? J Cereb Blood Flow Metab 14: 671–679. 34. Hochachka PW, Dunn JF (1983) Metabolic arrest: the most effective means of protecting tissues against hypoxia. Prog Clin Biol Res 136: 297–309. 35. Baugh LR, Hill AA, Brown EL, Hunter CP (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res 29: E29. 36. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386. PLoS Genetics | www.plosgenetics.org 11 October 2008 | Volume 4 | Issue 10 | e1000221