*2.16. Gene Expression Analysis*

The levels of gene expression of antioxidant enzymes (TrxR1, GR, NQO1 and SOD1) were evaluated with Real-Time PCR and β-actin was used as reference. Firstly, Caco-2 cells (5 × 105) were cultured into six well plates for 48 h in a complete medium and then treated with **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** (0.05 mg/mL) for 24 h. Cells were rinsed with 1 mL PBS 1×, lysed with 1 mL of TRIzol reagen<sup>t</sup> (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and transferred into a new test tube. At this point, 0.2 mL of chloroform were added and vigorously mixed for 15 s. Then samples were placed at RT for 15 min and subsequently centrifuged at 12,000× *g* for 15 min at 4 ◦C. In this way, three phases were obtained and the upper one, that contain mRNA, was transferred in a new test tube. Samples were added of 0.5 mL of isopropanol, mixed and maintained at RT for 10 min and subsequently centrifuged at 12,000× *g* for 10 min at 4 ◦C. The pellets were rinsed with 1 mL of 75% ethanol and mixed vigorously for 1 min. Samples were centrifuged at 7500× *g* for 10 min at 4 ◦C and the pellets were air-dried. The extracted mRNA was diluted in 0.01 mL of RNase free water and its concentration was estimated using NanoDrop system (Thermo Fisher Scientific). mRNA (1 μg) was subjected to reverse transcription using Euroscript M-MLV Reverse Transcriptase in the presence of 25 μg/mL oligo(dT), 10 mM DNTp mix, 10 mM DTT and RNase inhibitors (Euroclone, Milan, Italy) in a final volume of 0.02 mL. Before adding the reverse transcriptase, the mRNA was denatured at 65 ◦C for 5 min, then the mix were placed at 42 ◦C for 50 min and at 70 ◦C for 15 min. The resulting cDNA (1.5 ng/μL) was used for the Real-Time PCR analysis using Hot FIREpol Eva Green qPCR Supermix (Solis BioDyne, Tartu, Estonia). The target cDNA was amplified as follow: an initial step for polymerase activation at 95 ◦C for 12 min and then 40 cycles of denaturation for 15 s at 95 ◦C, annealing at 65 ◦C for 1 min and elongation at 72 ◦C for 1 min. All the primers were purchased from Sigma-Aldrich (St Louis, MO, USA). GR: Fw: 5-TCA CGC AGT TAC CAA AAG GAA A-3, Rv: 5-CAC ACC CAA GTC CCC TGC ATA T-3; TrxR1: Fw: 5-GCC CTG CAA GAC TCT CGA AAT TA-3, Rv: 5-GCC CAT AAG CAT TCT CAT AGA CGA-3; NQO1: Fw: 5-GGA GAC AGC CTC TTA CTT GCC AAG, Rv: 5-CCA GCC GTC AGC TAT TGT GGA TAC; β-actin: Fw: 5-ACC TGA CTG ACT ACC TCA TGA AGA-3, Rv: 5-GCG ACG TAG CAC AGC TTC TC-3; SOD 1: Fw: 5-TCA GGA GAC CAT TGC ATC ATT-3, Rv: 5-CGC TTT CCT GTC TTT GTA CTT TCT TC-3.
