Gsdmd n

Total protein was extracted from adenoid tissues and HNEpC cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Following mixing with SDS lysis buffer and boiling, the protein content in cell lysates were measured using BCA Protein Assay reagent (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were loaded onto 12% SDS-PAGE and separated via electrophoresis, then separated proteins were transferred to PVDF membranes. Following blocking in 5% skimmed milk at room temperature for 2 h, membranes were incubated overnight with primary antibodies against IL-32 (cat. no. ab37158; 1:1,000; Abcam), NLRP3 (cat. no. ab263899; 1:1,000; Abcam), IL-1β (cat. no. ab216995; 1:1,000; Abcam), caspase-1 (cat. no. ab207802; 1:1,000; Abcam), GSDMD-N (cat. no. ab215203; 1:1,000; Abcam), NOD1 (cat. no. ab189435; 1:1,000; Abcam), NOD2 (cat. no. ab31488; 1:500; Abcam), Toll-like receptor 4 (TLR4; cat. no. ab13556; 1:500; Abcam) and GAPDH (cat. no. ab8245; 1:5,000; Abcam). Membranes were then incubated with goat anti-rabbit IgG (cat. no. ab6721; Abcam; 1:2,000) or goat anti-mouse IgG (cat. no. ab6789; Abcam; 1:2,000) secondary antibodies at room temperature for 2 h. Proteins were then visualized using a gel imaging system (Amersham; Cytiva). Protein expression levels were semi-quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

The total protein of sciatic nerve tissue was extracted. First, the nucleus protein was extracted by nucleus protein extraction kit (Beyotime Bio, China). The content was detected by BCA protein detection kit (Thermo, Waltham, MA) according to the instructions. Then, the protein (30 µg/samples) with 10% SDS-PAGE was isolated and transferred to the PVDF membrane, sealed in 5% skim milk under 25°C for 1 h, and incubated with primary NF-B P65 (1:2000, Abcam Biotech, Cambridge, MA, USA), Caspase1 (1:1000, Abcam), Pro-Caspase1 (1:1000, Cusabiao, Wuhan, China), GSDMD-N (1:1000, Abcam), Klotho (1:1000, Abcam), NLRP3 (1:1000, Abcam), GAPDH (1:2500, Abcam) and H3 (1:2000, Abcam). And then, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 1 h. After that, protein bands were detected with a ECL detection kit (Beyotime Biothech, Shanghai, China), GAPDH or H3 was taken as control.

Cell culture was purchased from GIBCO (USA); The antibodies were as follows: NLRP3 (Cat#AG-20B-0014-C100), and ASC (AG-25B-0006A) were from Adipogen, San Diego, CA, USA, Caspase-1 (Cat. No.: 22915-1-AP) was from Proteintech, Chicago, USA, GSDMD-N (ab209845) was acquired from Abcam (Cambridge, USA); The Cell Whole Protein Extraction Kit and the BCA Protein Content Detection Kit were bought from KeyGEN BioTECH (Beijing, China). Calcein-AM and PI were purchased from Tongren Chemical (Beijing, China); IL-1β and IL-18 kits were purchased from Boster Biological Technology (Wuhan, China). Punicalin (purity 98.4%, batch, 20180706) was from Chengdu Herbpurify Co. Ltd (Chengdu, China); LPS, ATP, and N-Acetylcysteine (NAC) were bought from Netotime (Beijing, China). LPS and NAC were prepared as a 0.5 μg/mL stock solution in PBS (HyClone, USA), filter-sterilized, and stored at −20°C for up to two weeks.

Tissue sections (4 µm) were fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin for the following experiments. The sections were deparaffinized in xylene for 20 min and then washed with deionized water for 5 min. Then, the sections were treated with 10 nM citrate buffer and subsequently incubated with primary antibodies against Ki67 (1:1,000; cat. no. ab15580; Abcam) and GSDMD-N (cat. no. ab215203; Abcam). These slices were then incubated with the corresponding secondary antibody. Sections were observed and imaged under an inverted microscope (Leica Microsystems, Inc.).