A HT1080 OT-KD cells were reconstituted with Otulin WT or Otulin TM (TM represents the K64R, K197R, D31A mutant). Otulin WT and Otulin TM protein levels were assessed by Western blots, post-SeV infection (MOI 10) for the indicated time points. B IRF3 linear ubiquitination was analyzed in OT-KD cells reconstituted with either Otulin WT or Otulin TM mutant post-SeV infection of 24 h. Immunoprecipitation assay was performed by IRF3 antibody and immunoblotting was done by LUB9 antibody to detect IRF3 linear ubiquitination. C C-PARP levels were analyzed post-SeV infection of 24 h in HT1080 WT and HT1080 TM cells as a measure of apoptotic cell death. D SeV-C protein levels were assessed post-SeV infection in HT1080WT and HT1080 TM cells as a measure of viral protein synthesis. E HT1080 WT and HT1080 TM cells were infected with SeV for 24 h (MOI 10) and infectious viral yields were measured. Quantification of western blots and statistical analyses are provided in Supplementary Fig. 8 (mean ± SEM, n = 3; ns > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001).