Lentiviral shrna plasmids

The shRNA lentiviral plasmids were obtained from Sigma (St Louis, Missouri, USA). The shRNA against human MLKL (NM_152649.4) corresponds to the 3' untranslated region 2025–2045 (shMLKL1) and 1907–1927 (shMLKL2). CRISPR plasmids targeting human RIPK1 (NM_003804) and human RIPK3 (NM_006871) were generated according to Zhang’s protocol [42 (link)]. The sequence for CRISPR-RIPK1 was 5’-CACCGGATGCACGTGCTGAAAGCCG-3’ and CRISPR-RIPK3 was 5’-CAGTGTTCCGGGCGCAACAT-3’. All of the plasmid constructs were confirmed by DNA sequencing. To generate lentiviral particles, HEK293T were co-transfected with packaging plasmid (pCMV-VSV-G) and envelope plasmid (pCMV-dr8.2-dvpr) and either shRNA-non-targeting (shNT; pLKO.1puro) or shRNA-MLKL (shMLKL) or CRISPR-V2 or CRISPR-RIPK1 or CRISPR-RIPK3 plasmids. After 24 h, supernatants were collected and supernatants containing viral particles were filtered through a 0.45 μM sterile filter membrane (Merck Millipore, Darmstadt, Germany). The lentiviral preparation was then used to infect the cells with 8 μg/mL of polybrene (Merck Millipore, Darmstadt, Germany). After 24 h of infection, cells were selected with puromycin (Merck Millipore, Darmstadt, Germany) for a further 48 h.

The shRNA lentiviral plasmids were purchased from Sigma. The shRNA against mouse ZBP1 (NM_021394) or Noxa (NM_021451.1) corresponds to the 3′-untranslated region. The genomic DNA was isolated for PCR.