Figure 3

CATH-2 induces NLRP3 activation via a P2X7R-independent manner but dependent on K⁺ efflux in LPS-primed macrophages. Cells were primed with LPS for 3 h and then CATH-2 (5 μM) and ATP (1.5 mg/mL) as positive control were added for an additional 21 h. To inhibit K + efflux and the P2X7 receptor, cells were untreated (UT) or treated with KCl (5 mM and 50 mM) and the P2X7R inhibitor (A-740003, 100 μM) after LPS stimulation. Finally, cell supernatants and lysates were collected for different assays. ELISA was used to determine cytokine secretion of IL-1β (A, D) and TNF-α (B, E). A representative image (Western blot) is presented to show protein expression including IL-1β, caspase-1, pro-IL-1β, pro-caspase-1 and β-action (C, F). Data in A, B, D, E are represented as mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.