Rotor gene 1

The expressions of DNA repair-related genes were determined by using a real-time PCR kit, based on RealMOD™ SYBR Green (Intron, Seong-Nam, Korea), that employed Rotor Gene-Q (Qiagen, Valencia, CA) with gene-specific primer pairs (Additional file 1: Table S1). The PCR reactions were carried out at 95 °C for 5 min followed by 50 cycles of 95 °C for 5 s and 60 °C for 30 s, according to the manufacturer’s protocol. The threshold/quantification cycle (Ct/Cq) value was determined at the point in which fluorescence was detected statistically above the background, and PCR products were analyzed by generating a melting curve, constructed by Rotor-Gene 1.7 software (Qiagen, Valencia, CA). PCR was run in independent triplicates. The relative quantification of these gene expressions was calculated by the 2^−ddCt method.

Total RNA was extracted from cells using EuroGOLD trifast (Euroclone), according to the manufacturer’s instructions, and reverse-transcribed as previously described (24 (link)). qPCR was carried out using a Rotor-Gene 6000 (Corbett, Qiangen, Ancona, Italy) using iQ SYBR Green Supermix (Applied Biosystems, Milan, Italy). The sequences of the primers used for amplification of TataBox Binding Protein (TBP) housekeeping gene are Forward 5′-GAGCCAAGAGTGAAGAACAGTC-3′; Reverse 5′-GCTCCCCACCATATTCTGAATCT-3′. The sequences of SP-D primers are Forward 5′-AGGCTGCTTTCCTGAGCATGAC-3′; Reverse 5′-CCATTGGTGAAGATCTCCACACAG-3′. The melting curve was recorded between 55 and 99°C with a hold every 2 s. The relative amount of gene production in each sample was determined by the Comparative Quantification method supplied as part of the Rotor Gene 1.7 software (Corbett Research) (25 (link)). The relative amount of each gene was normalized with TBP and expressed as arbitrary units (AU) considering 1 AU obtained from fully differentiated macrophage used as calibrator.

RNA was purified from cells with EuroGOLD trifast (Euroclone) according to the manufacturer's instructions. Total RNA was extracted and reverse-transcribed as previously described [24 (link)]. Quantitative Real-Time PCR (qPCR) was carried out on a Rotor-Gene 6000 (Corbett, Qiagen, Ancona, Italy) using iQ SYBR Green Supermix (Bio-Rad, Milan, Italy). Table 1 shows the primer list used for qPCR. The melting curve was recorded between 55°C and 99°C with a hold every 2 s. The relative amount of gene production in each sample was determined by the Comparative Quantification (CQ) method supplied as part of the Rotor Gene 1.7 software (Corbett Research) [25 ]. The relative amount of each gene was normalized with 18S and expressed as arbitrary units (AU) considering 1 AU obtained from fully differentiated macrophage used as calibrator.

Cells RNA were purified with euroGOLD Total RNA Kit (Euroclone, Milan, Italy) according to the supplier’s instructions. miRNA isolation were carried out with miRNeasy Mini Kit (Qiagen, Milan, Italy) according to the supplier’s instructions. Total RNA extracted was reverse transcripted with iScript cDNA Synthesis Kit (Bio-Rad, Milan, Italy). Real-time quantitative PCR (qPCR) was carried out on a Rotor-Gene 6000 (Corbett, Explera, Ancona, Italy) using iQ SYBR Green Supermix (Thermo Scientific Fynnzymes, Milan, Italy). Table 1 show the primer lists used for qPCR. The relative amount of gene production in each sample was determined by the Comparative Quantification (CQ) method supplied as part of the Rotor Gene 1.7 software (Corbett Research)22 .

THP-1 cells (600,000/well) were seeded in 1 mL of medium containing 10 μg/mL of Phorbol 12-myristate 13- acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) in a 24-well flat-bottom plate (Costar, St. Louis, MO, USA) and were incubated for 2 days at 37 °C in 5% CO₂. Next, the PMA was removed and the cells were incubated o/n with fresh medium. Then the pro-inflammatory stimuli (10 ng/mL Ultrapure lipopolysaccharide LPS from E. coli 0111:B4 strain, Sigma-Aldrich USA, or 5000 U/mL IFN-γ, Sigma-Aldrich, USA) and/or the peptide were added to the cells and the plate was incubated for 24 h at 37 °C in 5% CO₂. After incubation the RNA extraction, cDNA synthesis, and the real-time quantitative PCR (RT-qPCR) were performed as previously reported [22 (link)]. The relative amount of gene production in each sample was determined by the Comparative Quantification (CQ) method supplied as part of the Rotor Gene 1.7 software (Corbett Research, Cambridge, UK) [23 (link)] normalizing the values with 18S and expressing as arbitrary units (AU) considering 1 AU obtained from resting macrophages as calibrator. Primer sequences are reported in Table 1.
The level of TNF-α in the supernatants collected from the blood macrophages was measured with commercial ELISA kits following the manufacturer’s instructions (Boster Immunoleader Tema Ricerca, Bologna, Italy).

RNA was extracted from cells with euroGOLDtrifast (Euroclone, Milan, Italy) according to the supplier’s instructions and reverse transcripted as previously described [13 (link)]. qPCR was carried out on a Rotor-Gene 6000 (Corbett, Explera, Ancona, Italy) using iQ SYBR Green Supermix (Bio-Rad, Milan, Italy). Supplementary Table S1 shows the primers used for RT-qPCR. The melting curve was recorded between 55 °C and 99 °C with a hold every 2s. The relative amount of gene expression in each sample was determined by the Comparative Quantification (CQ) method supplied as part of the Rotor Gene 1.7 software (Corbett Research) [35 ]. The relative amount of each gene was normalized with 18S and expressed as arbitrary units (AU) considering 1 AU obtained from decidual tissue used as a calibrator.