## 4 Materials and Methods
### Pathogen Adhesion Assay to Caco-2 Cells  
For the adhesion assay, Caco-2 cells were seeded and differentiated in 24-well plates (Becton Dickinson, Milan, Italy) at 1 \(\times\) 10\({}^{6}\) cells/well. Cells were placed in an antibiotic- and serum-free cell culture medium 16 h before the assay. On the day of the assay, overnight bacterial cultures of the pathogen indicator strains _L. monocytogenes_ OH and \(S\). Typhimurium LT2 were diluted 1:10 in appropriate media and grown for approximately 2 h up to the exponential growth phase. After monitoring the OD 600, bacterial cells were harvested by centrifugation at 5000\(\times\)\(g\) for 10 min, resuspended in antibiotic- and serum-free cell culture medium and added to the cell monolayers at a concentration of 1 \(\times\) 10\({}^{8}\) CFU/well (approximately 100:1 bacteria-to-cell ratio). CAR1 or THY5 were apically added together with the different indicator strains at a 0.02% concentration, identified as the highest nontoxic one from the Caco-2 permeability tests. Co-cultures of bacteria and Caco-2 cells were incubated at 37 \({}^{\circ}\)C for 1.5 h. Non-adhering bacteria were then removed by 5 washes with Hanks' Balanced Salt Solution (Corning) and cell monolayers were lysed with 1% Triton-X-100, according to [PERSON] et al. [62]. Adhering, viable bacterial cells were quantified by plating appropriate serial dilutions of Caco-2 lysates on different media. For \(S\). Typhimurium LT2, plating was performed by inclusion in violet red bile glucose agar (VRBGA) medium, while for _L. monocytogenes_, OH cell lysates were plated on Oxford medium. Plates were incubated for 18 h.  
Inflammation Induction by TNF-\(\alpha\) and EO Treatment for Gene Expression Analysis of NF-kB Pathway  
To assess the potential anti-inflammatory ability of CAR1 and THY5 EOs assayed in the present study, Caco-2 cells differentiated on Transwell filters were pre-incubated for 1 h with CAR1 or THY5 at the 0.02% concentration in the AP compartment. After media withdrawal, cells were challenged for an additional hour with the pro-inflammatory cytokine TNF-\(\alpha\) (Invitrogen, Rodano, Milan, Italy), added to both the AP and BL compartments at 15 ng/mL. Other samples were treated only with EOs for 1 h or only with TNF-\(\alpha\) for 1 h.  
At the end of treatments, RNA was extracted from the Caco-2 cells by using miRNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Nucleic acid concentration was determined by a NanoDrop(r) ND-2000 UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an OD of 260 nm. All samples had an OD 260/OD 280 ratio higher than 1.8, corresponding to 90-100% of pure RNA. A total of 1 ug of RNA was reverse-transcribed into cDNA using ReadyScript(tm) cDNA Synthesis Mix (Sigma-Aldrich). The quantification of gene expression was determined by real-time PCR with a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA), using RT\({}^{2}\) SYBR(r) Green ROX qPCR Mastermix (Qiagen). Data were collected using 7500 software v2.0.5 and given as the threshold cycle (Ct). Ct values for each target and housekeeping gene were obtained and their difference was calculated (\(\Delta\)Ct). Primer efficiencies for all tested genes were similar. The comparative calculation, \(\Delta\)\(\Delta\)Ct, was used to find the difference in the expression levels between the control and treated samples. Data are expressed as the mean of log2 of fold change (FC) with respect to the control.  
The following target genes were analyzed: inhibitor of nuclear factor kappa B (IkB)\(\alpha\), cellular inhibitor of apoptosis (cIAP)2, interleukin (IL)-1 \(\alpha\), IL-6, IL-8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Primer sequences are reported in Table S2.