Il 1β

First, equal amounts of protein were leaded in each lane of a polyacrylamide gel. Next, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C. The membrane was then washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated IgG. Finally, we employed ECL Western blotting detection reagents (Thermo Fisher Scientific) to visualize the protein bands and used Un-Scan-It 6.1 software (Silk Scientific, Orem, UT, USA) to measure the band density.

HNSCC cells (2 × 10⁵) were seeded in 24-well plates overnight. The next day, PBMCs (3 × 10⁶) were cocultured with the HNSCC cells with/without 10 µg/mL CDA for 2 and 6 hours. For transwell cocultures, 2 × 10⁵ HNSCC cells were seeded into the bottom compartment of a 24-well plate overnight. PBMCs (3 × 10⁶) were seeded into the top 0.4-µm ThinCert cell culture insert (Greiner Bio-One, catalog no. 665641) the next day and cultured for 6 hours ± 10 µg/mL CDA. Supernatants were harvested and the levels of IFNβ, IL6, TNFα, and IL1β were assessed with human type-I IFN (Biorbyt, catalog no. orb561974), TNFα (Invitrogen, catalog no. 88–7346), IL6 (Invitrogen, catalog no. 88–7066), and IL1β (Invitrogen, catalog no. 88–7261) ELISA kits following the manufacturer's instructions.