Nbp1 52813

Tissue sections were completely submerged in citrate buffer and repeatedly heated three times in a microwave oven. After natural cooling, the sections were washed and closed with 10% normal goat serum at RT for 30 min; after which, they were incubated in a wet box with diluted RORα antibody (NBP1‐52813, 1:400; Novus Biologicals) at 4°C for 12 h. After washing, the sections were incubated with biotin‐labeled goat anti‐rabbit antibody at RT for 30 min. The sections were then washed and subjected to DAB color development. Next, the sections were washed again, treated with DAB and hematoxylin, and sequentially processed for differentiation, dehydration, and transparency. After sealing, RORα expression in the tissues was observed under a microscope.

An aliquot from each group of suspended HNEpC cells (including crawling cells) was inoculated in the wells of a 24‐well plate at a density of 5 × 10⁴ cells/well. After being mounted the cells were washed and fixed with 40 mL/L paraformaldehyde for 30 min, and then permeabilized with 10 mL/L Triton X‐100 for 15 min. The cells were then blocked with 5 g/L bovine serum albumin at RT for 30 min and subsequently incubated with antibody of IL‐33 (12372‐1‐AP, 1:500; Proteintech), RORα (NBP1‐52813, 1:400; Novus Biologicals) or LC3B (PA01524, 1:300; Boster) at 4°C overnight. After washing, the cells were incubated with FITC‐labeled goat antirabbit IgG (1:100) for 60 min at 37°C. After DAPI (Sigma) staining of the nucleus, the cells were blocked with antiquenching agent and observed and photographed under a fluorescence microscope.

The nasal mucosa tissues from each group of mice were collected and ground to a powder. Aliquots of HNEpC and LAD2 cells that had been treated were collected. Total protein was extracted by using ice‐cold RIPA lysis buffer and the total protein concentration in each extract was determined by the BCA method. A 50 μg sample of protein from each extract was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the separated protein bands were transferred onto polyvinylidene fluoride membranes (Merck), which were subsequently blocked. Next, the membranes were incubated overnight at 4°C with primary antibodies of RORα (NBP1‐52813, 1:1000; Novus Biologicals), LC3 (PA01524, 1:1000; Boster), bECLin 1 (PB9076, 1:1000; Boster), p62 (PB0458, 1:2500; Boster), NLRP3 (27458‐1‐AP, 1:2000; Proteintech), Caspase 1 (81482‐1‐RR, 1:5000; Proteintech), ASC (10500‐1‐AP, 1:4000; Proteintech), and GAPDH (GB15002, 1:2000; Servicebio). After washing, the membranes were incubated with a secondary antibody at room temperature for 1.5 h. The immunostained protein bands were detected by enhanced chemiluminescence (ECL; Thermo). The Grayscale values of the target protein bands were analyzed using ImageJ software.