Figure 2.

CAPS-associated mutant NLRP3-induced necrotic cell death in the absence of pro-IL-1β. (a) WT and D301N-NLRP3-Tet-on-MC/9 cells were transfected with the pro-IL-1β-pMX-IP retroviral vector and incubated for 48 h. NLRP3-Tet-on-MC/9 cells (1 × 10⁶) were treated with 1 μg/ml doxycycline. Cells and supernatants were harvested for western blotting at the indicated time points after doxycycline treatment. (b) WT, D301N and Y570C-NLRP3-Tet-on-MC/9 cells (1 × 10⁶) were stimulated with 1 μg/ml doxycycline for 16 h. MC/9 cells were pretreated with 0.5 μg/ml LPS for 2 h and stimulated with 5 mM ATP for 45 min. The supernatants were harvested for immunoblotting of caspase-1. The indicate non-specific band. (c) Confocal scanning image of cell death caused by the expression of CAPS-associated mutant NLRP3, taken 6 h after doxycycline treatment. Scanning was performed at 1-min intervals with an Olympus FV10i. (d) Upper panels: electron microscopy of WT-NLRP3-Tet-on-MC/9 cells 10 h after doxycycline treatment. Lower panels: electron microscopy of D301N-NLRP3-Tet-on-MC/9 cells 10 h after doxycycline treatment. (e and f) WT and D301N-NLRP3-Tet-on-MC/9 cells were treated with 1 μg/ml doxycycline, and the signal intensities of EGFP and Alexa647-annexin-V/ 7-AAD staining were analyzed by flow cytometry. MC/9 cells were incubated with 20 μg/ml nigericin for 6 h as a necrosis control. (g) CAPS-associated mutant NLRP3-Tet-on-MC/9 cells (1 × 10⁶) were treated with 1 μg/ml doxycycline for the indicated time and the cells were harvested for immunoblotting of NLRP3, PARP, cleaved caspase-3 and actin. The asterisk indicates non-specific band. MC/9 cells were incubated with 100 nM actinomycin D for 8 h as an apoptosis control