immune function [40,66], cancer [67], aging [68], neurodegener- ative diseases [69,60] and adult neurogenesis [70]. Since thousands of Drosophila GAL4 lines have been characterized, this technique provides a method to non-invasively measure dCREB2 and NF-kB transcriptional activity in multiple different tissues. We believe this general approach can be used to study any transcription factor and its regulation in defined cell populations. Materials and Methods Fly Lines The following fly stocks were used: elav-GAL4, UAS-FLP1, hs- FLP, hs-GAL4, gmrlong-GAL4, actin-GAL4 (Bloomington Stock Center), ok107-GAL4 (kindly shared by Y. Zhong), repo-GAL4 (kindly shared by B. Jones), elavc155–GAL4 (kindly shared by K. Iijima-Ando), per0, perS, perL (kindly shared by A. Sehgal), and cg- GAL4 (kindly shared by B. Ganetzky). Reporter constructs The new CRE-F-luc reporter contains three copies of the symmetrical CRE site (59-TGACGTCA-39) situated upstream of a canonical TATAA element. A Cavener element [37] (CAAC) and an ATG translation initiation codon are situated downstream of where transcription initiates, followed by a cassette containing FRT sites flanking the mCherry gene (without its own ATG start codon). Translation that initiates at the ATG codon would continue through the FRT site into mCherry, and would terminate at the end of the mCherry coding sequence. A short sequence coding for a poly-glycine run is downstream of the second FRT site, and placed so that it is in the same reading frame as the ATG start codon, regardless of which FRT site remains after site-specific recombination. The luciferase-coding region (minus its normal ATG start codon) is placed downstream, and in frame with, the poly-glycine run. In the absence of FLP, the transgene would produce a fusion protein with amino acids encoded by one FRT sequence, and the mCherry open reading frame. After FLP-mediated recombination, a fusion protein would be encoded that contains amino acids from the FRT sequence and a poly-glycine run, all fused to luciferase. Sequence information is available upon request. The NFkB-F-luc reporter relies upon a kB element (59- GGGGACTTTCC-39) contained in the mammalian immuno- globulin/HIV promoter [71]. The design of the transgene is identical to the CRE-F-luc, except that the kB element is substituted for the CRE sites. Reporter constructs were synthe- sized (GenScript), sequenced, and inserted at the NotI/XhoI sites of pCaSper5. Standard methods were used to generate transgenic flies (BestGene). Luciferase Assays In vitro biochemical assays of luciferase activity were preformed on frozen fly homogenates as previously described [72]. For the head isolation experiment, three groups of 20 flies (10 male, 10 female) were frozen and heads and bodies were isolated and homogenized in cold homogenization buffer (15 mM HEPES, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA) [73]. Luciferase activity was measured in triplicate using the SteadyGlo Luciferase Assay System (Promega). In vivo luciferase assays were performed as previously described [13,27,28]. Briefly, 24–48 flies were entrained on a 12:12 light:dark cycle for 4–5 days and loaded into a black 96-well microplate containing luciferin media: 1% agar 5% sucrose food containing D-Luciferin (Gold BioTechnology). 5 mM luciferin was used for most of the reporter assays: (Fig. 1, 3A–D, S2, Table Oscillating dCREB2 and NF-kB Activity S1) or 25 mM luciferin was used for all others. Plates were maintained at 22uC under controlled light conditions and cycled approximately once per hour through a Packard TopCount Scintillation and Luminescence counter. For all experiments, plates were in the dark during detection periods (,20 min/hr). The first 12–24 hours after loading are excluded from analysis to allow flies to recover from handling. A smoothing function is applied such that each data point represents the average of three measurements. For comparisons of overall activity between groups, the mean hourly reading is calculated for each fly and compared using Student’s T-tests. For pre-feeding experiments, flies were maintained in groups of 50 flies on media containing luciferin (100 mM) for 24 h before being loaded individually into 96-well microplates plates contain- ing food alone (1% agar, 5% sucrose). The time delay between loading and the first luminescence measurement is between 30 m and 1 h. Drug Feeding Pyrrolidine dithiocarbamate (PDTC, Sigma) was mixed in yeast paste containing 15 mM luciferin. Flies were maintained in standard food vials coated in yeast paste for 24 h. After drug feeding, they were transferred to 96-well plates containing standard luciferin media for 1 h before luciferase activity was measured. Heat Shock For heat shock experiments, flies were maintained at 22u throughout development. Flies receiving heat shock were placed at 37u (air) for 40 minutes. Flies were allowed to recover for 24 h before all assays. Bacterial Infection Micrococcus luteus (gram-positive) and Escherichia coli (gram- negative) were cultured in LB medium overnight at 37uC. A mixture of these two types of bacteria was used for bacterial infection. A thin needle was dipped in a concentrated solution of bacteria and used to prick the thorax of adult flies (10,15-day old). Control flies received the same injury without bacteria. After infection, flies were loaded into a 96-well plate to monitor luciferase activity. Supporting Information Figure S1 Supplemental CRE-F-luc reporter validation. (A) CRE-F-luc reporter activity is FLP-dependent. Reporter activity (Y-axis, relative light units) is plotted as a function of genotype (singly [CRE-F-luc] or doubly transgenic flies [hs-FLP/ +;CRE-F-luc/+] and treatment (heat shock HS+ or not, HS2). The histogram bars indicate mean hourly counts over a 3-day window (n = 24). (B) Anatomical specificity of gmr-GAL4 driven reporter. In vitro luciferase activity measured in extracts made from dissected eye or remaining head tissue (n = 5). The relative light units (Y-axis) are plotted as a function of the genotype (shown in gray [UAS-FLP/+; CRE-F-luc/+ or black [UAS-FLP/gmrlong- GAL4; CRE-F-luc/+]) or tissue source. (Error bars = S.E.M, **** = p,0.0001). (TIF) Figure S2 NF-kB reporter activity oscillates over the day-night cycle. FLP-activated NFkB-F-luc activity. Singly (NFkB -F-luc) or doubly transgeneic flies (hs-FLP/+; NFkB -F- luc/+) are maintained under 12:12 LD conditions. Flies are exposed to heat-shock (+HS) or not (2HS), and measured for in PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e45130