Cells were seeded in 12-well plates at a density of 2.5 × 105 cells/well. Cells were collected and frozen in liquid nitrogen after treatment. Total RNA was extracted using a GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The concentration and purity of total RNA were examined using a Nanodrop 2000 (Thermo Fisher Scientific). Following digestion with DNase I, 1 μg of total RNA was reverse transcribed using a RevertAid cDNA synthesis Kit (Thermo Fisher Scientific). mRNA expression levels of NLRP3 and IL-1β were detected by real-time PCR using a HT7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The reaction mixture contained 3 μL cDNA template, 5 μL SYBR Green FastStart 2X Master Mix (Roche, Branford, CT, USA), and 0.3 μL gene-specific primers (Table (Table1).1). The program was composed of 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. A dissociation stage was added to check the amplicon specificity. This procedure was performed in accordance with our previously published method11. Relative expression levels were analysed using a comparative threshold cycle (2−∆∆Ct) method and normalised to GAPDH gene expression.

Primers used for real-time PCR.

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