Dmem glutamax

The strains used in this study are detailed in Table S1 in the supplemental material. The plasmids used in this study are described in Table S2. To induce T3SS expression, EHEC TUV93-0 was grown in minimal essential medium (MEM)-HEPES (Sigma, St. Louis, MO) with 5.62 g/liter of l-glutamine and EPEC was grown in Glutamax-Dulbecco modified Eagle medium (DMEM) (Sigma) at 37°C at 180 rpm. C. rodentium was also grown in Glutamax-DMEM (Sigma) and was cultured statically at 37°C with 5% CO₂. Growth and cell viability assays were carried out in lysogeny broth. For selection of plasmids, chloramphenicol or ampicillin was included at 20 or 50 μg/ml, respectively.

The cytotoxicity of the plant extracts was estimated on two different types of human colon cancer cells (HCT-116 and Caco-2). The test was performed as reported by Dawra et al. [26 (link)]. The cell growth was assessed by the MTT assay. MTT is a yellow water-soluble tetrazolium salt that is reduced by the mitochondrial dehydrogenases of intact cells to a purple formazan product. The cells were introduced in a 96-well plate at 3.10⁴ cells/well in a volume of 100 μL of a suitable culture medium. After that, 100 μL of the same culture medium containing the plant extract was added. The final concentration of the extract in each well was 50 μg/mL. The culture media used were, respectively, RPMI 1640 for the HCT-116 cells and Dulbecoo’s modified Eagle’s medium GlutaMAX (DMEM) for the Caco-2 cells (Sigma Aldrich, Saint Louis, Missouri, USA). Tamoxifen was used as a positive reference. The microplate was incubated at 37 °C for 48 h. The supernatant was then removed and 50 µL of the MTT solution was added, followed by an incubation of 40 min. The absorbance was measured at 605 nm. The inhibition percentage of the cells’ proliferation was calculated as follows: $\% \mathrm{INB}=100\times \left(\frac{Ablank-Asample}{Ablank}\right)$ .

The reagent grade chemicals and cell culture compounds used, namely Dulbecco’s Modified Eagle’s Medium (DMEM + GlutaMAX™), antibiotic solution (penicillin-streptomycin), non-essential amino acids (NEAAs), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), fungizone, trypsin/EDTA solutions, Phosphate Buffer Saline (PBS), Fetal Bovine Serum (FBS), methylthiazoltetrazolium salt (MTT) dye and dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water (resistivity <18 MW cm) was obtained by filtering tap water through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Standards of vit C (176.12 g/mol), vit E (430.71 g/mol), QUE (338.27 g/mol) and RSV (228.24 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein hydrolyzates based on hydrolyzed salmon fish protein with added vitamin C and vitamin D3 (product 1), hydrolyzed salmon and mackerel fish protein with added vitamin C and vitamin D3 (product 2), hydrolyzed salmon and blue whiting fish protein with added vitamin C and vitamin D3 (product 3) and flounder skin collagen were kindly provided by NOFIMA (Norway). Stock solutions of antioxidants vit E, QUE and RSV were freshly prepared in DMSO and stock solution of vit C was prepared in H₂O at appropriate working concentrations while maintained in darkness at 4 °C.