Lentiviral shRNA knockdown

Lentiviral plasmids encoding shRNAs targeting GFP (control; SHC005) and MAVS (06: TRCN0000149206; 45: TRCN0000148945) were obtained from the Sigma Mission library (Merck Darmstadt). Lentiviral particles for transduction were generated as follows: HEK293T cells were seeded in 6-well plates and the next day transfected with 1 µg psPAX2 packaging plasmid (Addgene 12260), 500 ng pMD2.G VSV-G envelope plasmid (Addgene 12259) and 1 µg shRNA plasmid using Fugene 6 (Promega). The next day, the medium was replaced. After another 24 h, lentivirus-containing supernatant was harvested three times every 8–16 h. Pooled supernatants were filtered through 0.45 µm filters and stored at − 80 °C. Calu-3 cells were transduced by addition of lentiviral supernatants containing 8 µg/ml polybrene (Merck Darmstadt). 48 h after transduction, cells were selected by addition of media containing 5 µg/ml puromycin (Gibco). Surviving cells were used for experiments.