*2.13. ROS Production Estimation*

ROS production in Caco-2 cell line was measured by using 5-(and 6)-chloromethyl-20,70- dichlorohydrofluorescein diacetate (CM-H2DCFDA,Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells (1 × 104) were grown in a 96-wells plate for 48 h, and then treated with synthetic peptides (0.05 mg/mL) for 24 h. Cells washed in PBS 1×/10 mM glucose were loaded with 10 μM CM-H2DCFDA for 20 min in the dark at 37 ◦C. Subsequently, the fluorescent probe was removed and cells were rinsed with PBS 1×/10 mM glucose and subjected to oxidative stress in the presence of 250 μM TbOOH. Fluorescence increase was followed at 485 nm (λ excitation) and 527 nm (λ emission) for 90 min using a plate reader (Tecan Infinite ® M200 PRO, Männedorf, Switzerland).

## *2.14. Nrf2 Translocation to the Nucleus*

In order to investigate the Keap1/Nrf2 activation, the translocation of Nrf2 to the nucleus was followed. For this purpose, nuclear and cytosolic fractions were divided according to the method described by Yao et al. (2014), with some modifications [27,28]. Briefly, Caco-2 cells (1 × 106) were grown in T25 flasks for 48 h and then treated with 0.05 mg/mL **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E.** After 24 h, cells were rinsed with 1 mL of PBS 1× and lysed for 15 min on ice with 100 μL of buffer containing 10 mM Hepes/Tris pH 7.9, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 10 mM KCl, 1 mM NaF and a protease inhibitor cocktail (Complete, Roche®, Basel, Switzerland). The samples were rapidly added of IGEPAL (5% final concentration), mixed for 15 s and centrifuged at 1000× *g* for 10 min at 4 ◦C. The pellet (nuclear fraction) was dissolved in 20 mM Hepes/Tris (pH 7.9), 1 mM EGTA, 1 mM EDTA, 0.4 M NaCl in the presence of 0.1 mM PMSF, 1 mM NaF and protease inhibitors (Complete, Roche®, Basel, Switzerland). Samples were mixed every 2 min for 10–15 s and centrifuged at 20,000× *g* for 10 min at 4 ◦C to discard the debris. Nuclear proteins (30 μg evaluated according to Lowry et al. [29]) were subjected to SDS-PAGE (10%) and subsequently to western blot analysis to define the expression level of Nrf2. Densitometric analysis of WB was carried out using NineAlliance software (Mini 9 17.01 version, Uvitec Alliance, Cambridge, UK). PCNA was used as loading reference.
