FIGURE 4.

PML-deficient BMDM display enhanced levels of IL-1β and IL-18 in response to NLRP3 and AIM2 inflammasome activation. Primary BMDM from 129Sv wild type (WT) and PML^−/− mice were left unstimulated or stimulated with 100 ng/ml LPS for 3 h alone with or without the addition of 5 mm ATP for 1 h or transfected with 1 μg/ml poly(dA-dT) for 5 h as indicated. Supernatants were analyzed by ELISA for IL-1β (A), TNFα (B), and IL-18 (C) or by Western blotting for pro-IL-1β and the mature subunit IL-1β p17 (D). Densitometric analysis of IL-1β p17 levels in WT versus PML-deficient cells is also presented (right panel). BMDMs were also assessed for PML deficiency by Western blot (E). A representative blot from one of three independent experiments is shown (D and E). Relative mRNA expression of caspase-1 in WT and PML^−/− primary BMDMs in response to specific NLRP3 and AIM2 activation is also shown (F). RNA was isolated and the quantity of caspase-1 analyzed by real time PCR. A–C, results are expressed as mean ± S.D. for triplicate determinations. Results are representative of three individual experiments, ***, p < 0.001; **, p < 0.01; *, p < 0.05 PML^−/− versus WT controls (CTL).