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. 2017 Jul 17;36(17):2529–2543. doi: 10.15252/embj.201796476

Figure 5. High levels of ectopic ZBP1 expression sensitise cells to necroptosis.

Figure 5

  1. NIH3T3 cells or immortalised Zbp1 −/− MEFs expressing wild‐type or mutant ZBP1 were treated with 20 μM zVAD alone, 30 ng/ml TNF, or with TZ or were infected as indicated (MOI = 10). Cell viability was assessed as in Fig 1B after 16 h.
  2. NIH3T3 cells reconstituted with wild‐type or mutant ZBP1 were treated with 20 μM zVAD alone or TZ in combination with 3 μM GSK'872 (RIPK3 inhibitor) or 30 μM Nec‐1 (RIPK1 inhibitor) or 0.1% DMSO as a control. Cell viability was monitored after 24 h as in Fig 1B.
  3. Polyclonal and monoclonal NIH3T3 cell lines expressing no, low or high levels of ZBP1 were analysed by Western blot using the indicated antibodies. Asterisk (*) indicates a non‐specific band.
  4. Clonal cell lines from (C) were analysed as in (A).
  5. Zbp1 −/−‐immortalised MEFs expressing wild‐type or mutant ZBP1 were transduced with empty or with M36 lentiviral vectors. Cell numbers were determined after 4 days.
  6. Cells from (E) were tested by Western blot using the indicated antibodies.
Data information: Data are representative of two or more independent experiments. Panels (A, B, D and E) show mean ± SD (n = 3). *P < 0.05, ***P < 0.001; two‐way ANOVA. See also Fig EV5. Source data are available online for this figure.