Fibroblast transfection and stimulation

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.