Figure EV4. ZBP1 binds newly synthesised RNA in MCMV‐infected cells (related to Fig 4).

• A
  NIH3T3 cells (top panel) or immortalised Zbp1 ^−/− MEFs (lower panel) were treated with indicated concentrations of ganciclovir (GCV) and infected with MCMV‐M45^mutRHIM or UV‐C inactivated MCMV‐M45^mutRHIM as indicated. After 5 days, cells were fixed and stained with crystal violet.
• B
  RNA samples from the experiment shown in Fig 4B and C were analysed by RT qPCR.
• C
  Structure of the MCMV IE1/3 gene. Four different siRNAs specifically targeting IE3 (exon 5) were designed.
• D
  NIH3T3 cells were transfected with the indicated siRNAs and were then infected with MCMV‐IE1/3‐GFP. GFP‐expressing cells were enumerated using Incucyte analysis.
• E
  NIH3T3 cells expressing wild‐type or mutant ZBP1 were infected with MCMV‐M45^mutRHIM (MOI 3) and 3 h post‐infection 30 ng/ml TNF was added to the cells. Cell death was analysed as in Fig 4F.
• F
  NIH3T3 cells expressing wild‐type ZBP1 were transfected with the indicated siRNAs, infected with MCMV‐M45^mutRHIM (MOI 3) and 3 h post‐infection 30 ng/ml TNF was added to the cells. Cell death was analysed as in Fig 4F.
• G, H
  Immortalised Zbp1 ^−/− MEFs reconstituted with wild‐type ZBP1 were infected with MCMV‐M45^mutRHIM (MOI = 10) in the presence of neutralising anti‐IFNAR1 or isotype control antibodies. (G) 16 h post‐infection viability was assessed as in Fig 1B. (H) RNA samples were collected 8 h after infection and analysed by RT–qPCR.

Data information: Data are representative of two or more independent experiments. Panels (B, D–H) show mean ± SD (n = 3). *P < 0.05; one‐way ANOVA.