Fig 9. SiiD independently suppressed NLRP3 inflammasome activation by preventing mtROS-ASC signaling.

J774A.1 cells were transduced with Lv-SiiD or Lv-NC lentivirus, untreated cells were used as a negative control (Blank). Cells were primed with LPS (1 μg/mL) for 5 h, MitoQ (10 nM) or vehicle control (DMSO) was added to cells 1 h after LPS treatment. The cells were then stimulated with or without ATP (1.25 mM) or nigericin (10 μM) for 1 h. (A, E) Cells were lysed and the pellets were subjected to cross-linking. The activation of Caspase-1 (p10), the expression of SiiD, the ASC oligomerization in the pellets, and the total ASC in lysates as the input were examined by western blotting. β-actin was blotted as a loading control. Molecular mass markers in kDa are indicated on the right. (B, F) Supernatants were analysed for cytotoxicity evaluated by LDH release. (C, G) IL-1β and (D, H) IL-6 secretion in supernatants were examined via ELISA. Data are presented as mean ± SEM of triplicate samples per experimental condition from three independent experiments. **p < 0.01, ***p < 0.001; NS, not significant, as measured by one-way ANOVA followed by Bonferroni’s multiple comparison test.