*4.6. Measurement of Cytokine Release*

The amounts of IL-1β and IL-18 produced by J774.1 cells were measured using enzyme-linked immunosorbent assay (ELISA). Briefly, cells were seeded into a 24-well plate (1 × 10<sup>6</sup> cells/well) and incubated for 12 h in a 37 ◦C and 5% CO2 incubator. Subsequently, cells were treated with 2.5, 5, and 10 μM of juglone for 2 h, followed by LPS treatment (1 μg/mL) for 6 h and ATP treatment (5 mM) for 1 h. The production of pro-inflammatory cytokines was assessed using commercial ELISA kits (mouse IL-1β: R&D Systems, Minneapolis, MN, USA; mouse IL-18: Cloud-Clone Co., Katy, TX, USA) according to the manufacturer's instructions. The absorbance was measured at 540 nm. All experiments were performed in triplicate.

#### *4.7. RNA Isolation and cDNA Synthesis*

Total cellular RNA from J774.1 cells was isolated using the RNeasy Midi Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Samples were treated with RNase-free DNase I (Takara, Dalian, China) at 37 ◦C for 30 min to avoid any DNA contamination. The quality and quantity of the RNA samples were measured using a bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). RNAs with RNA integrity values ≥ 8.0 were used. Complementary DNA was synthesized from 1 μg total RNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). cDNA synthesis was performed at 46 ◦C for 20 min, followed by enzyme inactivation at 95 ◦C for 1 min.

#### *4.8. Real-Time Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)*

Total RNA was extracted using TRIzol reagen<sup>t</sup> (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RT-qPCR was performed on a CFX96 Real-time PCR (Bio-Rad Laboratories Inc., Hercules, CA, USA). RT-qPCR reactions were prepared using 1 μL cDNA, 10 μL SYBR Green master mix (Bio-Rad Laboratories, Inc.), and 2 μL of primer mix in a total volume of 20 μL. Thermocycling conditions were as follows: 3 min at 95 ◦C, 35 cycles of denaturation (15 s at 95 ◦C), and combined annealing/extension (30 s at 60 ◦C). GAPDH was used as a housekeeping gene. Primer sequences and their respective PCR fragment lengths were as follows: NLRP3 (NACHT, LRR and PYD domains-containing protein 3), forward 5--GTGGAGATCCT AGGTTTCTCTG-3- and reverse 5--CAGGATCTCATTCTCTTGGATC-3-; Caspase-1, forward 5--GAGCTGATGTT GACCTCAGAG-3- and reverse 5--CTGTCAGAAGTCTTGTGCTCTG-3-; IL-1β, forward 5--GTGGAGATCCTAGGTTTCTCTG-3- and reverse 5--CAGGATCTCATTCTCTTGGATC-3-; IL-18, forward 5--AGGACACTTTCTTGCTTGCC-3-, and reverse 5--CACAAACCCTCCCCACCTAA-3-; GAPDH forward 5--GTGGGGCGCCCCAGGCACCA-3- and reverse 5--CTCCTTAATGTCA CGCACGATTTC-3-. After amplification, a melting curve (0.01 ◦C/s) was used to confirm product purity. Relative mRNA content was normalized to GAPDH content.
