Figure 5. Pannexin‐1 is required for BAX and BAK‐mediated activation of NLRP3 but is dispensable for NLRP3 inflammasome assembly downstream of caspase‐8.

• A, B
  BMDMs of the indicated genotypes were seeded at a density of 2 × 10⁶ cells per well and were primed with 100 ng/ml LPS for 3 h and then treated with Cp. A (1 μM) for 6 h (A) or with 5Z‐7 oxozeaenol (TAK1i, 250 nM) or ABT‐737 (1 μM) and CHX (20 μg/ml) for 6 h, or nigericin (10 μM) for 45 min (B). Cell lysates and supernatants were analysed by western blot and the PBS‐insoluble fraction of the cell lysate was cross‐linked and assessed for ASC oligomerisation by western blot. Ponceau staining depicts protein loading. Data representative of two independent experiments.
• C
  BMDMs of the indicated genotypes were seeded at a density of 5 × 10⁵ cells per well and were treated as outlined in (A) and (B). Cell viability was determined by PI staining and flow cytometry and measured as a proportion of PI‐negative (live) cells. Data represent the mean of three to four independent experiments (symbols). Error bars are the mean ± SD.
• D
  BMDMs of the indicated genotypes were seeded at a density of 5 × 10⁵ cells per well and cell lysates were analysed by western blot.
• E–G
  IncuCyte live cell imaging analysis of BMDM death kinetics. BMDMs were seeded at a density of 7.5 × 10⁴ per well and primed with LPS (100 ng/ml) for 3 h before treatment with Cp.A (1 μΜ, E), TAK1i (250 nM, F) or ABT‐737 (1 μΜ) + CHX (20 μg/ml, G) for 14 h. Cell death was measured as a percentage of cytox green positive cells versus SPY620‐DNA positive cells. Each graph is representative of three independent experiments, data points represent the mean of triplicate wells. Error bars are the mean ± SD.

Source data are available online for this figure.