Keratinocyte transduction

HEK293T were cultured in high-glucose (4,500 mg/liter) DMEM (GIBCO BRL; 41965–039) supplemented with 10% FCS and 2 mM glutamine (Lonza; BE17-605F). For lentivirus production, HEK293T cells were transfected with N-terminally 3XFLAG and V5-tagged wild-type mouse ZBP1 or Zα1α2 mutant mouse ZBP1 transducing vectors in the pLenti6 backbone (Life Technologies; Maelfait et al., 2017) together with the pCMV delta R8.91 gag-pol–expressing packaging plasmids and pMD2.G VSV-G–expressing envelope plasmid. 24 h after transfection, cells were washed and FAD medium was added. 48 h after transfection, the viral supernatant was harvested and used to transduce 25,000 mouse primary keratinocytes seeded in 48-well plates in the presence of 8 µg/ml polybrene (Sigma-Aldrich). The next day, viral particle–containing medium was removed, and cell death was measured for 48 h as described above.