Figure 5.

NLRP3 inflammasome agonist nigericin induces apoptosis in a caspase-8-dependent manner. (a) Nigericin-induced cell death is delayed in Casp1^−/− BMMs. BMMs of the indicated genotypes were primed for 4 h with 10 ng/ml LPS prior to treatment for 1–6 h with nigericin at doses 0, 2.5, 5 and 10 μM. The degree of cell death was determined by PI staining followed by flow cytometry. The results shown are the means±S.E. of the data of three independent experiments. (b) Nigericin causes pyroptotic death of WT cells and apoptotic death of Casp1^−/− BMMs. BMMs were treated with 10 ng/ml LPS, followed by 10 μM nigericin for 2 h. Annexin-V–PI staining was used to detect apoptotic cells by flow cytometry. (c) Apoptotic death of Casp1^−/− BMMs was detected by the presence of sub-G₀/G₁ DNA content. Cells were primed with 10 ng/ml LPS for 4 h, followed by nigericin for 6 h. Each data point shows the result of an independent experiment. (d) Nigericin activates apoptotic caspases in an ASC-dependent and caspase-1-independent manner. BMMs from C57BL/6 (WT), Asc^−/− and Casp1^−/− mice were pretreated with 10 ng/ml LPS and after 4 h nigericin was added to the cells at a final concentration of 10 μM. Control cells were treated with a methanol vehicle control. Cell protein extracts and proteins released into the culture medium were collected after 2.5 h of incubation at 37 °C, and analysed by western blot. (e) Caspase-8 is the apical apoptotic caspase activated by nigericin. Casp1^−/− iBMMs with caspase-8 or -9 knocked down were treated with 100 ng/ml LPS for 4 h followed by 10 μM nigericin for 75 min. Cell extracts were analysed by western blot. The degree of knockdown in this experiment is shown in Figure 4g. (f) Nigericin-dependent apoptosis requires caspase-8. Casp1^−/− iBMMs with caspase-8 or -9 knocked down were treated with 10 ng/ml LPS for 4 h followed by 10 μM nigericin for 4 h. The results shown are the mean and range of duplicate cell treatments from a single experiment. An effect of caspase-8 knockdown was shown in four independent experiments. The left-hand panel shows cell death measured by flow cytometry of PI-stained cells; the middle panel shows sub-G₀/G₁ cells assessed by flow cytometry as an indication of apoptosis; and the right hand panel shows the degree of caspase-8 and -9 knockdown, assessed by western blot. Example primary data from flow cytometry are shown in Supplementary Figure 4