Type 1 collagen coated plates

MoDCs were seeded at 1 × 10⁷ cells/12-well type I collagen-coated plates (Iwaki, Tokyo, Japan) and cultured for 5 days and stimulated with TLR2, TLR4, NOD1, or NOD2 ligands for 12 h. MoDCs (1 × 10⁷ cells/12 well) were also stimulated with the two paraimmunobiotic bifidobacteria individually (5 × 10⁷ CFU/mL) for 6h. PIE cells were seeded at 3 × 10⁴ cells/12-well type I collagen-coated plates (Iwaki, Tokyo, Japan), cultured for 5 days and then stimulated with TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, or NOD2 ligands for 12 h (Supplementary Table S3). PIE cells were stimulated with the two paraimmunobiotic bifidobacteria individually (5 × 10⁷ CFU/mL) for 6 h. In another set of experiments, PIE cells were seeded at 3 × 10⁴ cells/12-well plate on type I collagen-coated plates (Iwaki), cultured for 3 days and treated with paraimmunobiotic bifidobacteria (5 × 10⁷ cells/mL) for 48 h. Then, each well was washed vigorously with medium at least three times to eliminate bacteria, and cells were stimulated with different PRRs ligands (TLR2, TLR4, NOD2) for 12 h. The mRNA expression of PGLYRPs was evaluated by qRT-PCR.

In atorvastatin (Sigma-Aldrich, St Louis, Mo) cytotoxicity experiment, HepaRG (5 × 10⁵ cells/well), Flp-In-293/ABCB1 (2677G), Flp-In-293/ABCB1 (2677 T) and Flp-In-293/ABCB1 (2677A) cells were cultured in monolayers at 37 °C for 24 h in 24-well collagen type I-coated plates (Iwaki Glass, Chiba, Japan). After the preculture, cells were cultured in the presence of different concentrations of atorvastatin 0, 0.3, 1, 3, 10, 30, 100 and 300 μM for HepaRG cells, 0, 0.6, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mM for Flp-In-293/ABCB1 (2677G/T/A) cells for 24 h. After the culture, 50 μl of WST-8 working solution (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) was added to each well and the plates were incubated at 37 °C for 1 h under 5 % CO₂ and 95 % air. Optical density at 450 nm was measured by a microplate reader (ARVOmx, PerkinElmer, Waltham, MA). Lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and ALT releases from HepaRG cells into the medium were determined according to the manufacturers’ protocol of LDH, AST and ALT activity assay kits (Biovision, Milpitas, CA).