Fig 2: Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6 clones showed different NF-κB activation (a) and IL-8 production (b) in response to LAB. Caco-2-NF-κB-hrGFP C3 cells were seeded at 1 × 105 cell/well, while HT-29-NF-κB-hrGFP E5 and F6 clones were seeded at 5 × 104 cell/well, cultured ON, and treated for 2 h with L. reuteri ATCC 23272 or L. plantarum ATCC 8014. Then, gentamycin and TNF-α (1 ng/mL) were added; 48 h or 18–24 h later (for Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6, resp.), cells were trypsinized and NF-κB activation (measured by the % GFP+ cells) was analyzed by flow cytometry. Cell viability was over 90% for all tested conditions. Data was normalized against TNF-α control (considered as 100%). IL-8 quantification was performed in the harvested cell culture supernatant by flow cytometry. Results were expressed as the mean ± SD of triplicates of a representative experiment. * P < 0.05 using One-Way ANOVA with Dunnett's posttest.

Fig 3: MTT assay results for compounds 17, 20, 21 and 22. MTT HT29: human colon adenocarcinoma cell line (ATCC No. HTB-38) MTT test results; MTT CCD 841 CoTr: human normal colon epithelial cells (ATCC No. CRL-1807) MTT test results.

Fig 4: Pre-incubation with L. reuteri ATCC PTA 6475 and ATCC 53608 inhibits EPEC adherence to HT-29 but not LS174T cells. (A) HT-29 cells were pre-incubated with a 100-fold excess of L. reuteri ATCC PTA 6475, ATCC 53608, or left untreated (–) for 1 or 3 h before addition of EPEC for 1 h. (B) HT-29 cells were pre-incubated with a 100-fold (100x) or 10-fold (10x) excess or equal numbers (1x) of L. reuteri ATCC PTA 6475, ATCC 53608, or left untreated (–) for 1 h before addition of EPEC for 1 h. (C) LS174T cells were pre-incubated with a 100-fold excess of ATCC PTA 6475, ATCC 53608 or left untreated (–) for 1 h before addition of EPEC for 1 h. Cell-associated EPEC were quantified by CFU, and adhesion is expressed as percentage of adherent bacteria relative to the inoculum. Data are shown as means ± SEM from three independent experiments performed in duplicate. ∗P < 0.05, ∗∗P < 0.01.

Fig 5: Mucus binding proteins CmbA and MUB increase L. reuteri adhesion to HT-29 and LS174T cells. (A) Immunofluorescence staining demonstrating MUC2 expression (green) by LS174T but not HT-29 cells. Cell nuclei were labeled in blue. Shown are representative images from three independent experiments performed in duplicate. Bar = 10 μm. (B) HT-29 and LS174T cells were incubated with EPEC, or L. reuteri strains ATCC PTA 6475 or ATCC 53608 for 1 h. In addition to wild-type strains (WT), isogenic mutants deficient in expression of the adhesin CmbA (ATCC PTA 6475 CmbA-) or MUB (ATCC 53608 MUB-) were included for comparison. Adhesion is expressed as percentage of adherent bacteria relative to the inoculum. Data are shown as means ± SEM from three independent experiments performed in duplicate. ∗∗P < 0.01.