vivo luminescence. The relative luminescence is plotted as a function of time, with daytime (white bars) and nighttime (black bars) conditions indicated below the graph. Each data point represents the average of 24 flies. (TIF) Figure S3 PDTC inhibition of NFkB-F-luc reporter activity persists for 24 hours at high doses. The relative luminescence is plotted with respect to the PDTC dose fed to flies. Triply transgenic flies (UAS-FLP/+;NFkB-F-luc/actin-GAL4) were fed different dosages of PDTC for 24 hours and then measured for luminescence 1 h after the end of feeding. Reporter activity is pooled over 1 day following 24 h PDTC feeding (n = 24 for each group) (Error bars = S.E.M, *p,.05). (TIF) Figure S4 Neuronal CRE-F-luc reporter cycling is modulated in per mutants. Neuronal reporter activity is plotted over time as flies are shifted from light:dark to constant darkness. For all of these panels, the same transgenes (UAS-FLP/ +; NFkB-F-luc/elav-GAL4) exist in all flies, but the flies are examined in a wild type (A), per0 (B), perS (C) or perL (D) genetic background. (TIF) Figure S5 Oscillations in CRE-luc reporter activity persist after substrate removal. (A) Reporter activity (in References 1. Reid KJ, McGee-Koch LL, Zee PC (2011) Cognition in circadian rhythm sleep disorders. Prog Brain Res 190: 3–20. 2. Froy O (2011) Circadian rhythms, aging, and life span in mammals. Physiology (Bethesda) 26: 225–235. 3. Allada R, Chung BY (2010) Circadian organization of behavior and physiology in Drosophila. Annu Rev Physiol 72: 605–624. 4. Claridge-Chang A, Wijnen H, Naef F, Boothroyd C, Rajewsky N, et al. (2001) Circadian regulation of gene expression systems in the Drosophila head. Neuron 32: 657–671. 5. McDonald MJ, Rosbash M (2001) Microarray analysis and organization of circadian gene expression in Drosophila. Cell 107: 567–578. 6. Lin Y, Han M, Shimada B, Wang L, Gibler TM, et al. (2002) Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster. Proc Natl Acad Sci U S A 99: 9562– 9567. 7. Ceriani MF, Hogenesch JB, Yanovsky M, Panda S, Straume M, et al. (2002) Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J Neurosci 22: 9305–9319. 8. Ueda HR, Matsumoto A, Kawamura M, Iino M, Tanimura T, et al. (2002) Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J Biol Chem 277: 14048–14052. 9. Wijnen H, Naef F, Boothroyd C, Claridge-Chang A, Young MW (2006) Control of daily transcript oscillations in Drosophila by light and the circadian clock. PLoS Genet 2: e39. 10. Boothroyd CE, Wijnen H, Naef F, Saez L, Young MW (2007) Integration of light and temperature in the regulation of circadian gene expression in Drosophila. PLoS Genet 3: e54. 11. Keegan KP, Pradhan S, Wang JP, Allada R (2007) Meta-analysis of Drosophila circadian microarray studies identifies a novel set of rhythmically expressed genes. PLoS Comput Biol 3: e208. 12. Boothroyd CE, Young MW (2008) The in(put)s and out(put)s of the Drosophila circadian clock. Ann N Y Acad Sci 1129: 350–357. 13. Belvin MP, Zhou H, Yin JC (1999) The Drosophila dCREB2 gene affects the circadian clock. Neuron 22: 777–787. 14. Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, et al. (2010) Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Nat Med 16: 1152–1156. 15. Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, et al. (1993) Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260: 238–241. 16. Obrietan K, Impey S, Smith D, Athos J, Storm DR (1999) Circadian regulation of cAMP response element-mediated gene expression in the suprachiasmatic nuclei. J Biol Chem 274: 17748–17756. 17. Gau D, Lemberger T, von Gall C, Kretz O, Le Minh N, et al. (2002) Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock. Neuron 34: 245–253. Oscillating dCREB2 and NF-kB Activity relative light units, RLU) in CRE-luc reporter flies is plotted as a function of time, starting 1 h after the transfer from luciferin to non-luciferin food. Light conditions are indicated by white boxes (daytime) and grey boxes (nighttime). (B) The same data as in (A), with the first day excluded. (TIF) Methods S1 (DOC) Table S1 (DOC) Acknowledgments dCREB2 Reporter Driver Screen. We would like to thank the following people: Erin Gonzales worked on the original design of the FLP reporter, and Hong Zhou assisted in DNA work. Stefanie Tanenhaus and Dana Ballard wrote a program to import luminometer data. Stanislava Chtarbanova-Rudloff provided assistance with the bacterial infection. Kristin Widmer, Sara Anderson, and Jason Swanson provided assistance with the in vivo luciferase assays. Author Contributions Conceived and designed the experiments: AT JZ JY. Performed the experiments: AT JZ TT. Analyzed the data: AT JZ JY. Contributed reagents/materials/analysis tools: AT JZ TT JY. Wrote the paper: AT JZ JY. 18. Tischkau SA, Mitchell JW, Tyan SH, Buchanan GF, Gillette MU (2003) Ca2+/ cAMP response element-binding protein (CREB)-dependent activation of Per1 is required for light-induced signaling in the suprachiasmatic nucleus circadian clock. J Biol Chem 278: 718–723. 19. Lee B, Li A, Hansen KF, Cao R, Yoon JH, et al. (2010) CREB influences timing and entrainment of the SCN circadian clock. J Biol Rhythms 25: 410–420. 20. O’Neill JS, Maywood ES, Chesham JE, Takahashi JS, Hastings MH (2008) cAMP-dependent signaling as a core component of the mammalian circadian pacemaker. Science 320: 949–953. 21. Atkinson SE, Maywood ES, Chesham JE, Wozny C, Colwell CS, et al. (2011) Cyclic AMP signaling control of action potential firing rate and molecular circadian pacemaking in the suprachiasmatic nucleus. J Biol Rhythms 26: 210– 220. 22. Doi M, Ishida A, Miyake A, Sato M, Komatsu R, et al. (2011) Circadian regulation of intracellular G-protein signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus. Nat Commun 2: 327. 23. Marpegan L, Bekinschtein TA, Freudenthal R, Rubio MF, Ferreyra GA, et al. (2004) Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters. Neurosci Lett 358: 9–12. 24. Cecon E, Fernandes PA, Pinato L, Ferreira ZS, Markus RP (2010) Daily variation of constitutively activated nuclear factor kappa B (NFKB) in rat pineal gland. Chronobiol Int 27: 52–67. 25. Lee JH, Sancar A (2011) Regulation of apoptosis by the circadian clock through NF-kappaB signaling. Proc Natl Acad Sci U S A 108: 12036–12041. 26. Kuo TH, Pike DH, Beizaeipour Z, Williams JA (2010) Sleep triggered by an immune response in Drosophila is regulated by the circadian clock and requires the NFkappaB Relish. BMC Neurosci 11: 17. 27. Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, et al. (1996) Novel features of drosophila period Transcription revealed by real-time luciferase reporting. Neuron 16: 687–692. 28. Stanewsky R, Jamison CF, Plautz JD, Kay SA, Hall JC (1997) Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J 16: 5006–5018. 29. Asai M, Yamaguchi S, Isejima H, Jonouchi M, Moriya T, et al. (2001) Visualization of mPer1 transcription in vitro: NMDA induces a rapid phase shift of mPer1 gene in cultured SCN. Curr Biol 11: 1524–1527. 30. Wilsbacher LD, Yamazaki S, Herzog ED, Song EJ, Radcliffe LA, et al. (2002) Photic and circadian expression of luciferase in mPeriod1-luc transgenic mice invivo. Proc Natl Acad Sci U S A 99: 489–494. 31. Yamaguchi S, Isejima H, Matsuo T, Okura R, Yagita K, et al. (2003) Synchronization of cellular clocks in the suprachiasmatic nucleus. Science 302: 1408–1412. 32. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, et al. (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science 288: 682– 685. 33. Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, et al. (2004) LUCIFERASE real-time reporting of circadian dynamics reveals persistent PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e45130