Heat inactivated fbs

Human primary dermal fibroblasts (of the first 8 passages; ±2 passage difference across cell lines of different individuals) that were genetically screened for MIRAS point mutations (by DNA-seq) were used for analyses. Fibroblasts were cultured in DMEM (Lonza; with 4.5 g l⁻¹ glucose) supplemented with 10% (v/v) heat-inactivated FBS (Lonza), 50 U ml⁻¹ penicillin–streptomycin (Gibco), 0.05 mg ml⁻¹ uridine (Calbiochem) and 2 mM GlutaMAX (Gibco) at 37 °C under 5% CO₂, with fresh medium replaced every 2 days, and were tested negative for mycoplasma. Transfection of synthetic dsDNA^{50 (link)} and dsRNA (poly(I:C), Sigma-Aldrich) was performed using FuGENE HD transfection reagent (Promega). In brief, around 2 × 10⁵ cells were plated onto six-well dishes the day before transfection and transfected with 2.5 μg of dsDNA or dsRNA per well with a 1:2 ratio of nucleic acid:transfection reagent, according to the manufacturer’s instructions (sequence details are provided in Supplementary Table 6). For expression of RIG-I or MAVS, fibroblasts were transfected with pcDNA3.1(+)-Flag containing RIG-I (N) or MAVS^{51 (link)} before poly(I:C) transfection 24 h later and incubated for another 7 h before collection.

The HT29-MTX mucus-secreting subpopulation of human colon carcinoma cell line HT29 was kindly provided to Muriel Thomas (Micalis, INRA, Jouy-en-Josas, France) by Dr. Thécla Lesuffleur (INSERM UMR S 938, Paris, France) (Lesuffleur et al. 1990 (link)). The HT29-MTX cells were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) containing phenol red and 4.5 g l⁻¹ glucose (Lonza, Verviers, Belgium), supplemented with: 10% (v/v) heat-inactivated FBS (Lonza, Verviers, Belgium), 1% (v/v) l-glutamine 200 mM (Lonza, Verviers, Belgium) and 1% (v/v) penicillin-streptomycin mixture (10,000 U ml⁻¹ and 10,000 μg ml⁻¹, respectively) (Lonza, Verviers, Belgium). The cells were seeded at a concentration of 2.5 × 10⁴ cells cm⁻² in six-well tissue culture plates (Thermo Fisher Scientific, Nunc A/S, Waltham, MA USA) or on glass coverslips placed in 24-well tissue culture plates (Thermo Fisher Scientific, Nunc A/S, Waltham, MA USA) for adhesion experiments and for fluorescent staining experiments, respectively. Two days before the experiments, antibiotics were no longer added to the cell culture medium. HT29-MTX cells were used between passages 27 and 46. To ensure full differentiation of cells, experiments were carried out 20 to 22 days post seeding. The cells were maintained at 37 °C in a humidified atmosphere with 10% CO₂, and the culture medium was changed daily.

A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza). BHK-21 were also supplemented with 20% Tryptose Phosphate Broth (18050039, Thermo Scientific). HEK-293T cells were supplemented with sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM). HEK-Blue IFN-α/β cell line (InvivoGen) was maintained in HEK-293T media supplemented with 10μg/ml blasticidin (InvivoGen) and 200 μg/ml Zeocin (InvivoGen). Vero E6 cells (Cercopithecus aethiops kidney epithelial cells, ATCC CCL-81) were cultured in Minimum Eagle Medium (MEM) (11095–080, Gibco), supplemented with 2 mM L-glutamine, 50 U/mL penicillin-streptomycin and 7.5% heat-inactivated FBS. A549, BHK-21, HEK-293T and Vero E6 cell lines were originally provided by J.C. de la Torre (The Scripps Research Institute, La Jolla, CA) and HEK-Blue IFN-α/β cell line by S. Sharma (La Jolla Institute for Allergy and Immunology, La Jolla, CA)