Fresh human leukapheresis products were obtained from the EFS (Marseille, France). Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-PaqueTM PLUS (Amersham Biosciences, Uppsala, Sweden), washed four times with RPMI and CD14+ cells were immunomagnetically purified with AutoMACS system following the protocol of the manufacturer (Miltenyi Biotech, Auburn, CA). Purified CD14+ monocytes were analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA), confirming the purity of CD14+ cells to be 95%. To promote differentiation into iDC, the purified CD14+ cells (0.5 × 106 cells/ml) were plated in 6-well plates (2 × 106 cells/well) and cultured in RPMI 1640 medium supplemented with 10% FCS, non essential amino acids, penicillin/streptomycin 100 ng/mL (>1000 U/ml), recombinant human GM-CSF and 20 ng/mL (>100 U/ml) IL-4 for 5 days (both from PeproTech, Rocky Hill, NJ). At days 2 and 4, half of the volume of the medium was replaced by fresh medium supplemented with GM-CSF and IL-4. For DC maturation, 100 ng/mL LPS (Escherichia coli type 026:B6; Sigma-Aldrich, St. Louis, MO) was added to the cells at day 5, for the indicated number of hours.
Polysomal profiling by sucrose gradient fractionation
Polysome-bound mRNA molecules were enriched by sucrose gradient fractionation following the protocol originally developed by Garcia-Sanz and collaborators . Briefly, 60 to 80 × 106 day 5 human moDCs were lysed in 1 ml of polysome buffer (10 mM Tris-HCl (pH 8), 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP40, 0.1 mg/ml cycloheximide, and 500 units/mL RNasin (Promega, Madison, WI). After 10 min on ice, lysates were quickly centrifuged (10.000 × g for 10 sec at 4°C) and the supernatant was resuspended in a stabilizing solution (0.2 mg/ml cycloheximide, 0.7 mg/ml heparin, 1 mM phenylmethanesulfonyl fluoride). After a quick centrifugation (12.000 × g for 2 min at 4°C) to remove mitochondria and membrane debris, the resulting supernatant was layered on a 15% to 40% sucrose gradient. Gradients were then ultracentrifuged (35.000 × g for 2 h at 4°C, SW41 rotor) and after centrifugation 20 × 550 ml fractions were collected, starting from the top of the gradient. All the fractions were then digested with Proteinase K (200 mg/ml) in presence of 1%SDS and 10 mM EDTA. RNA was then extracted with Phenol/Chloroform/Isoamylalcohol (volume ratio 25:24:1) and precipitated with 2.5 Volumes of 100% Ethanol in presence of 0.8 M lithium chloride, necessary to get rid of heparin, a known inhibitor of RT activity . After precipitation all the RNA were resuspended in 20 ml RNase free H2O. The correct fractionation of the polysomes was tested by detecting the different rRNA types on a 1% denaturing agarose gel. Total RNA was directly extracted out of moDCs without fractionation. Total and polysomal-bound RNAwere purified using the RNeasy miniprep kit (Qiagen, Chatsworth, CA). To exclude theamplification of genomic DNA, an on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen). The RNA Integrity Number (RIN-value) of all RNA types and timepoints was measured with the Agilent 2100 Bioanalyzer. RIN-values between 6.5 and 8.5 (mean RIN value = 7.6) were obtained, indicating that the RNA had sufficient integrity to be analyzed by microarrays.
Affymetrix microarray hybridization and data mining
For each condition 100 ng of total or polysomal-bound RNA were employed to synthesize double-stranded cDNA using two successive reverse-transcription reactions according to standard Affymetrix protocols (GeneChip Two-Cycle Target Labelling, Affymetrix, Santa Clara, CA). Linear amplification with T7-RNA polymerase and biotin labelling were performed by in vitro transcription by standard Affymetrix procedures. The resulting biotin-labeled cRNA was fragmented and hybridized to the Affymetrix Human Genome U133 2.0 oligonucleotide 14,500-gene microarray chip for 16 h at 45°C. Following hybridization, the probe array was washed and stained on a fluidics station and immediately scanned on a Affymetrix GCS 3000 GeneArray Scanner. The data generated from the scan were then analyzed using the MicroArray Suite software (MAS 5.0, Affymetrix). The data derived from four independent experiments were normalized using the GC-RMA algorithm and bioinformatic analysis was performed using GeneSpring GX 7.3 (Agilent, Palo Alto, CA) and Statistics Analysis System (SAS v9.1.3). Probe selection was performed using 2-way ANOVAs accounting for repeated measures with a false discovery rate of 0.05. Hierarchical clustering was performed using the default clustering algorithm and setting in GX7.3.
Quantitative real-time RT-PCR
Total RNA was extracted and purified using the RNeasy kit (Qiagen). To exclude the amplification of genomic DNA, an on-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen). 1 μg of RNA was retro-transcribed using SuperScript II reverse transcriptase (Invitrogen) and random (pDN6) primers. First-strand cDNA templates were then used for PCR amplification of short (100 to 150 bp) exon fragments of the gene of interest using the appropriate primers (Additional file 4 shows the complete list of the 375 probe sets with statistically significant interaction). PCR was carried out using a Stratagene MX3000P Real-Time PCR System in complete SYBR Green PCR buffer (PE Biosystems, Warrington, UK) using 200 nM of each specific primer. A total of 20 μl of PCR mix was added to 5 μl of cDNA template, and the amplification was tracked via SYBR Green incorporation by using a Stratagene (La Jolla, CA) sequence detection system 4.01. Comparative real-time PCR was done in triplicate, including no-template controls. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified. Relative quantification of target cDNA was determined by calculating the difference in cross-threshold (C
t) values after normalization to GAPDH signals, according to the Pfaffl method and the automated Excel-based program available (REST©). The sequences of all employed primers are available in Additional file 7.
Immunodetection and antibodies
50 μg of TX-100 soluble material or 200.000 cells, lysed directly in Laemmli buffer, were loaded on 10% SDS-PAGE prior to immunoblotting and chemiluminescence detection (SuperSignal, Pierce, USA). SUnSET was performed as previously described  using A647-labelled mouse IgG2a anti-puromycin antibody (12D10). Antibody against RPL26 was from Abnova. For flow cytometry analysis, APC-conjugated anti-HLA-DR (L243 clone) and PE-conjugated anti-CD86 (clone IT2.2) were from BD PharMingen (San Jose, CA, USA). Anti- eIF2, phospho-eIF2 and -phospho-S6 were from Cell Signaling Technology (Beverly, MA, USA). For immunofluorescence analysis, MoDCs were let adhere on Alcyan blue coated-coverslips and surface stained with unlabelled antibody at 4°C for 30 min. After washes, coverslips were placed in warm medium for the indicated time and then fixed in 3% PFA for conventional staining with secondary antibodies.