Cycloheximide (CHX), chlorophenylthio-cAMP (cpt-cAMP) and glucagon-like peptide-1 (GLP-1) (7–37, human) were purchased from Sigma (Buchs, Switzerland).
Min6 cell culture and incubations
Min6 B1 cells  (generously provided by Dr. Philippe Halban, Dept of Development and Medical Genetics, Medical faculty of Geneva University) (passage 15–25) were cultured in DMEM supplemented with 15% fetal calf serum, 25 mM glucose, 71 μM 2-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamycin. Medium was changed to low glucose medium (same as above with 1% FCS and 1 mM glucose) 20 hours before a 4 hour stimulation with glucose (10 mM) and cpt-cAMP (0.2 mM). When used, CHX (5 μg/ml) was added 45' prior to stimulation.
Islet and primary beta cell isolation, culture and incubations
7-Week-old male Wistar rats (~250 g) were purchased from Elevage Janvier (Le Genest-St-Isle, France). Pancreatic islets were isolated by collagenase digestion, handpicked, and maintained in 11.1 mM glucose/RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (Amimed, BioConcept Allschwil, Switzerland), 100 units/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamycin (Sigma).
For the induction experiments, islets were maintained for 48 hours after isolation and serum deprived for 20 hours in low glucose medium (1 mM glucose RPMI 1640 (Invitrogen), 0.1% BSA and same antibiotics as above). Consecutively, islets were stimulated with glucose, GLP-1 or cpt-cAMP as detailed in the respective figure legends.
For beta cell purification, islets were trypsin digested and FACS sorted as earlier described . Beta cells were maintained in 11.1 mM glucose/Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum and same antibiotics as above, for five days to permit re-aggregation in small clusters. 20 hours before stimulation cells were serum deprived in low glucose medium (1 mM glucose/DMEM supplemented with 0.1% BSA, and same antibiotics as above).
RNA preparation and microarray analysis
All the stimulations were performed at the same time (in parallel), with an unique batch of cells plated at uniform confluence. For each experimental condition, transcript profiles were established for three different preparations of total RNA made using RNeasy Micro Kit (Qiagen). Labeled cRNA synthesis, hybridization to the arrays and scanning were essentially performed as earlier described . Affymetrix Mouse Genome 430 2.0 oligonucleotide array (containing probe features for 45'101 transcripts) were used. Fluorescence signals from the arrays were analyzed with Affymetrix software GCOS for normalization and calculation of gene-expression values [38, 39].
Criteria to define differentially expressed transcripts
The definition of differentially expressed transcripts between two experimental conditions was based on three criteria: concordance between replicates, statistical significance and fold-change cutoff. The strategy to evaluate the concordance of an effect in two different experimental conditions was the following: each replicate of one condition was compared to each replicate of the other, resulting in 9 pairwise comparisons. Transcripts were considered as differentially expressed if their levels changed in the same direction in at least 7/9 comparisons. The second criterion was a p value by Welch T test below 0.05 when comparing signal values in two experimental conditions. Finally, the third criterion was a minimal fold-change values of 1.5.
Criteria to delineate IEGs and targets
We considered genes that were differentially expressed in control (no stimulation) vs stimulated (glucose + cAMP) condition; these representing glucose and cAMP regulated genes. We divided this list of genes into two lists: IEGs and targets. To do so, different criteria were used.
The main criterion was the responsiveness in presence of CHX. From the initial list of regulated genes, we considered genes that were differentially expressed in control+CHX (no stimulation in presence of CHX) vs stimulated+CHX (glucose + cAMP in presence of CHX) conditions; these represent IEGs (genes that respond to glucose and cAMP in presence of CHX). The rest of the genes (that were initially found to respond to glucose and cAMP but were not regulated in presence of CHX) were defined as targets. This single criterion is very stringent for the definition of IEGs, but may lead, on the other hand, to a high level of false positive in the target category. This is particularly unsuitable, notably for the validity of target promoter sequence analysis. Thus it was necessary to introduce more criteria to increase the quality of target list.
Criteria to increase target list quality (secondary criteria)
Some genes responded to CHX alone. If CHX produces more effect than glucose, it can mask the effect of glucose in presence of CHX (saturating regulation by CHX). For this reason, IEGs can be falsely considered as targets with the main criterion (here-above). Thus we excluded from up-regulated target gene list, the genes for which expression was higher in either control+CHX or stimulated+CHX conditions compared to the stimulated condition. Similarly, we excluded from down-regulated target gene list, the genes for which expression was lower in either control+CHX or stimulated+CHX conditions compared to the stimulated condition. Finally, we excluded from the target list, genes for which the mean signal difference between stimulated+CHX and control represented more than 25% of the signal in the stimulated condition. Genes excluded from the target list by these secondary criteria were attributed to the IEG list.
Quantitative real-time RT-PCR
Each total RNA sample was reverse-transcribed in triplicate with random hexamers as primers and Omniscript reverse transcriptase (Qiagen). Quantitative real-time PCR were performed with the SYBR Green system as described in Brun et al. . Primers were provided by Microsynth (Balgach, Switzerland) and their sequences are presented in Additional file 9. For normalization, 18S RNA was quantified in each sample using 0.3× 18S rRNA Predeveloped Assay Reagent and 1× TaqMan® Universal PCR Master Mix (Applied Biosystems).
PCR amplicons were quality controlled and all displayed a single homogeneous melting curve as well as the correct size on 2% agarose gels. A cDNA serial dilution standard curve was added to the microtiter plate of each amplification reaction to calibrate each relative quantification in function of PCR amplification efficiency.
TFExplorer predicted regulatory element database  was used to map regulatory elements in promoters (from -1000 bp to +300 bp from transcription start site) (accessed on June 17th 2005 ). We analyzed promoters of target genes (132 up-regulated gene promoters, 239 down-regulated gene promoters) and of two control sets of promoters from genes randomly chosen among those present (detectable in Min6 cells, 1188 promoters) or those absent (undetectable in Min6 cells, 1164 promoters). For each promoter set (up-regulated targets, down-regulated targets and controls) we counted the number of promoters (Hit numbers) in which a given regulatory element was present (at least once). We calculated the frequencies for any given regulatory element within each set, and evaluated the statistical significance of the difference to the control sets by Fisher exact test.
Nuclear extract preparation and DNA binding assay
Nuclear protein extracts were prepared according to the protocol of Schreiber et al.. The detection of c-FOS and JUND specific binding to AP-1 site was made with the ELISA-like TransFactor Kit Inflammation II (BD Biosciences AG, Switzerland) according to supplier instructions except that the colorimetric detection step was replaced by a chemiluminescent one. Briefly, after initial blocking, 12 μg of nuclear extracts were incubated 60 minutes in AP-1 or STAT consensus oligo coated 96-well plates. Plates were then washed three times, incubated 60 minutes with primary antibodies (anti-c-FOS or anti-JUND), washed three times and incubated 30 minutes with HRPO-anti-rabbit-IgG secondary antibody (Transduction Laboratories) (1:10'000). After final four washes, 100 μl of 1× ECL HRP substrate (Cell Signaling Technology) were added to each well and light emission measured three times with a FLUOStar OPTIMA (BMG LABTECH GmbH). Binding to coated STAT oligo and competition with soluble AP-1 oligo were used to check binding specificity. Results were expressed in arbitrary units of DNA binding after normalization by values of no template controls (NTC) for each independent experiment.
Srxn1 reporter construction
Srxn1 promoter regions of three different sizes (-421/+39; -109/+39; -28/+39 from the transcriptional start site) were amplified by PCR. Primer were designed from sequences found in ENSEMBL database (entry: ENSMUST00000041500) with addition of 5' flanking residues to create restriction sites (XhoI for forward primers, HindIII for the reverse primer; allowing directional insertion). Three different forward primers were used srxn1-421, AACTCGAG AGACAGCGCTGGGATCCAA; srxn1-109, AACTCGAG GGCCTGAGTCACCACGCT; srxn1-28, AACTCGAG CGTCCATTGAGCGCATCG (XhoI site in bold). A single reverse primer was used srxn1+39: GATTAAGCTT CTGACCTAGCTGCCCACTGCC (HindIII site in bold). PCR products were initially cloned into pGEMT-easy vector (Promega) using Takara mighty mix DNA ligation kit (Takara Bio Inc.) and sequentially restriction digested with HindIII and XhoI (Roche). Inserts of respective expected sizes were cloned into pGL3enhancer vector (Promega) that had been previously restriction digested with the same enzymes and treated with alkaline phosphatase (Roche). Construction sequences were verified by the Dye Terminator sequencing technique using Rvprimer3 (CTAGCAAAATAGGCTGTCCC) at the DNA sequencing facility of Geneva University Medical Center.
Luciferase reporter analysis
0.5 μg PathDetect® cis-Reporting System pAP-1-Luc or pCIS CK (negative control) plasmids (Stratagene Europe, Amsterdam Zuidoost, The Netherlands) were co-transfected with 0.5 μg of Renilla luciferase plasmid (for normalization) (Promega, Luzern, Switzerland) using Lipofectamine 2000 reagent (Invitrogen) according to supplier's instructions. In the ectopic expression experiment, pMSCV-c-Fos (c-Fos expression vector) and/or pMSCV-c-JunFlag (c-Jun expression vector)  (both generously provided by Dr. Gerald Thiel, University of Saarland Medical Center, Germany) were cotransfected at various concentrations (see figure legends). Luciferase activity measurement was performed 24 hours after the transfection as previously described . In stimulation experiments, cells were transfected with reporter vectors, maintained for 20 hours in culture medium, changed to low glucose medium for additional 20 hours, and stimulated for 6 hours with 10 mM glucose and 0.2 mM cpt-cAMP (in triplicate).
AP-1 loss-of-function experiments
pCMV500 (control), pCMV500-A-FOS and pCMW500-A-C/EBP (kind gift of Dr. Charles Vinson, National Cancer Institute, Laboratory of Metabolism, Bethesda, MD, USA) were used in transient co-transfection with reporter constructs or for establishment of stable transfectant Min6 clones. In the latter case, after transfection, 400 mg/l G418 were added to culture medium for a selection period of one month. Clones were picked-up and grown in culture medium supplemented with 200 mg/l G418. A decrease in AP-1 reporter was specifically found in A-FOS clones transiently transfected with pAP-1-luc (Additional file 6). At least three different clones for each construct were used in the experiments.
Western blotting and immunocytochemistry
Nuclear extracts (15 μg) were resolved on SDS-PAGE (10% gel) and subject to immunoblot analysis as earlier described . Primary antibodies were rabbit anti-c-FOS (1:1'000, sc-52) and anti-TFIIB (1:10'000, sc-225) (Santa Cruz Biotechnology, Inc.). For immunofluorescence studies, partially trypsin dispersed rat islets were cultivated and pre-incubated in low glucose RPMI 1640 medium as described above for intact islets. After stimulation, cells were subjected to cytospin on SuperFrost®Plus slides (Menzel GmbH and Co KG, Braunschweig, Germany) and fixed in 4% paraformaldehyde pH 7.0 for 30 minutes at room temperature. After three PBS washes and two incubations with boiling 10 mM citrate pH 6.0 for two minutes, cells were permeabilized with 0.2% Triton PBS for 15 minutes. Primary antibody for c-FOS (rabbit anti-c-FOS, 1:200, sc-52, Santa-Cruz Biotechnology) and mouse anti-insulin (1:1000, I-2018, Sigma) were diluted in 0.05% triton PBS and used for an overnight incubation. After three washes, cells were incubated one hour with secondary antibodies (alexa-488 labeled anti-rabbit-IgG and alexa-568 labeled anti-mouse-IgG (both 1:300; Molecular Probes)). After washings, cells were incubated three minutes in 5 mg/ml 4',6-Diamidino-2-phenylindol (DAPI), washed three times and mounted in Dakocytomation fluorescent mounting medium (DakoCytomation AG, Untermüli, Switzerland). Images were acquired with a Zeiss Axiocam Imaging System (Bioimaging Core Facility, Medical Faculty, Geneva University).
Numbers of repeated experiments
In the figure legends, n represents the number of repeated experiments. This corresponds to different cell preparations (in the case of islets, preparation from different rats).