Antibodies against nf κb p65

EVO (Natural Institutes for Food and Drug Control, Beijing, China) was dissolved in complete medium containing 0.2% dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St Louis, MO, USA) as a 100-mM stock solution and stored at −20°C. GEM (Eli Lilly and Company, Indianapolis, IN, USA) was reconstituted as a 200-µg/mL stock solution in sterile normal saline (NS) and stored at −20°C. Antibodies against NF-κB p65 were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-Bcl-2, anti-Bcl-xl, and anti-β-actin antibodies were obtained from Immune Way (Plano, TX, USA). Human Cal-27 (American Type Culture Collection [ATCC], Manassas, VA, USA) and Tca-8113 (Medical University of the Air Force, Xian, China; approved by the medical ethics committee of Lanzhou University) tongue squamous carcinoma cell lines were cultured in DMEM, supplemented with 10% FBS, and maintained in a humidified atmosphere with 5% CO₂ at 37°C. Male nude BALB/c mice aged 4–5 weeks old (18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). Mice were housed in specific pathogen-free laboratory of Gansu University of Chinese Medicine (Lanzhou, China).

The cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. The antibodies against GSDMD (1:1,000; cat. no. ab209845), caspase-1 (p20) (1:500; cat. no. ab1872), IL-1β (1:1,000; cat. no. ab9722), autophagy-related (ATG)-5 (1:1,000; cat. no. ab199560), Beclin-1 (1:1,000; cat. no. ab62557), heme oxygenase-1 (HO-1; 1:1,000; cat. no. ab137749) and β-actin (1:1,000; cat. no. ab8227) were purchased from Abcam. The antibodies against NLRP3 (1:1,000; cat. no. 13158), P62 (1:1,000; cat. no. 13121S), and LC3B (1:1,000; cat. no. 2775) were obtained from Cell Signaling Technology, Inc. The antibodies against NF-κB p65 (1:1,000; cat. no. 14220-1) and Nrf-2 (1:1,000; cat. no. 16396-1) were purchased from ProteinTech Group, Inc., and the antibodies against phosphorylated (p)-NF-κB p65 (Ser536) (1:1,000; cat. no. AF2006) and Nucleolin (1:1,000; cat. no. DF12542) were purchased from Affinity Biosciences. DI was purchased from Sigma-Aldrich; Merck KGaA. DAPI and other reagents were obtained from Beyotime Institute of Biotechnology.

Betaine (molecular formula: C₅H₁₁NO₂, CAS No.: 107-43-7, purity >98%) was procured from Shanghai Yuanye Bio-Technology Co., Ltd, China. AβO was purchased from China, Peptides Co. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Sigma-Aldrich, USA. The NF-κB activator phorbol 12-myristate 13-acetate [19 (link)] (PMA, a protein kinase C activator that can induce NF-κB activation in BV2 cells; this concentration was confirmed in previous studies; molecular formula: C36H56O8, CAS No.: 16561-29-8, purity >98%) was procured from MedChemExpress, China. A protein quantification (BCA assay) kit was provided by Beyotime Institute of Biotechnology, China. Antibodies against NF-κB p65, NLRP3, ASC, and Caspase-1 were provided by Cell Signaling Technology (USA). Enzyme-linked immunosorbent assay (ELISA) kits were procured from CUSABIO Technology, China (IL-1β ELISA kit: detection range: 31.25 pg/mL-2000 pg/mL; IL-18 ELISA kit: detection range: 1.56 pg/mL-100 pg/mL; TNF-α ELISA kit: detection range: 15.6 pg/mL-1000 pg/mL. Kit intra-assay precision: CV% <8%, interassay precision: CV% <10%).