Fig. 3: MafB knockdown promotes the activation of the NLRP3 inflammasome.

a–c PMs were transfected with 50 nM control or MafB siRNAs. Forty-eight hours after transfection, cells were treated with 100 ng/ml LPS for 3 h. Cells were then washed and stimulated with or without ATP (5 mM), Nigericin (5 uM) or MSU (1 mM) in serum-free media for an additional 1 h. Levels of the supernatant IL-1β (a) and IL-18 (b) were determined by ELISA. Levels of the supernatant LDH (c) were determined by LDH assay. d PMs were transfected, primed and stimulated with the NLRP3 agonists as in (a). Supernatant proteins were precipitated by TCA and combined with the respective cellular extracts. Levels of the indicated proteins were determined by western blotting. Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. e, f PMs were transfected with control siRNAs or MafB siRNAs. Forty-eight hours after transfection, cells were primed with LPS for 3 h, followed by stimulation with or without Nigericin for 1 h. Cells were then fixed with 4% PFA and subjected to immunofluorescence staining for ASC. Scale bars: 100 μm (e). ASC specks in the cells were quantified (f). n = 3–8. g, h J774A.1 macrophages were transfected with control siRNAs or MafB siRNAs. 48 h after transfection, cells were primed with LPS for 4 h, followed by stimulation with or without ATP (5 mM) or Nigericin (10 μM) for 1 h. Levels of the supernatant IL-1β and LDH were determined (g). Levels of the indicated proteins in the supernatants and cells were determined by western blotting (h). Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. Representative of three independent experiments. LDH lactate dehydrogenase.