Fig. 6: MafB restricts the NLRP3 agonists induced mitochondrial damage.

a, b J774A.1 macrophages were transfected with control siRNAs or MafB siRNAs. 48 h later, cells were treated with or without 100 ng/ml LPS for 4 h, ATP (5 mM) or Nigericin (10 µM) for 30 min, and stained with 100 nM MitoTracker Deep Red and 100 nM MitoTracker Green for additional 20 min. Cells were collected, and flow cytometric analysis was performed to determine the mitochondria damage. c Primed J774A.1 macrophages transfected with control siRNAs or MafB siRNAs were incubated with 2.5 µM MitoSOX for 15 min, followed by stimulation with ATP or Nigericin for 30–60 min. Mitochondrial ROS (mtROS) was determined by fluorescence microplate reader under 530/590 nm (Ex/Em). d, e J774A.1 macrophages transfected with control siRNAs or MafB siRNAs were primed with LPS for 4 h, followed by stimulation with Nigericin for 30 min. The cells were permeabilized with 25 µg/ml Digitonin for 10–15 min and cytoplasmic DNAs were prepared. Levels of mtDNA were determined by real-time PCR assay. f, g J774A.1 macrophages transfected with control siRNAs or MafB siRNAs were treated with LPS for 4 h, followed by MitoTempo (100 µg/ml) treatment for 30 min. The cells were stimulated with Nigericin for 60 min. Supernatant IL-1β was determined by ELISA (f), and levels of the indicated proteins in the supernatants and cells were determined by western blotting (g). Vertically stacked bands originate either from a single membrane or from a replicate membrane, with the same sample loadings. Mean ± SD; **P < 0.01, ***P < 0.001. Representative of three independent experiments. ns not significant.