Chemiluminescence system

Tissue or collected cells were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride and extracted. The protein concentration of the supernatant was determined using a spectrophotometer (Beckman, Fullerton, CA, USA). Proteins were separated by polyacrylamide gel electrophoresis, and they were transferred to a polyvinylidene fluoride (PVDF) membrane by electroblotting. The PVDF membrane was placed in a closed solution containing 5% (w/v) skim milk for 1 h, incubated with antibodies (1:1000–2000) for 6 h at 4°C, and then washed thrice with PBS and incubated with secondary antibodies (horseradish peroxidase-conjugated) for 1 h at 37°C. The PVDF membrane was soaked in an ECL working solution (Millipore, MO, USA) for color development. Finally, the proteins were detected, and images were captured using the chemiluminescence system (Bio-Rad Laboratories).

After trypsinization of the cells, proteins were extracted using radioimmunoprecipitation (RIPA) buffer (Beyotime Institute of Biotechnology, China) containing protease inhibitors (Thermo Fisher Scientific Technology, USA), and quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Total proteins were separated by SDS-PAGE on a 10% gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Sigma, USA). Following blocking with 5% skim milk at room temperature for 2 hours, primary antibodies against IGF2BP3 (1:1000; Abcam Institute, United States), METTL3 (1:1000, Abcam, United States), or CDK6(1:1000; Abcam Institute, United States) were incubated overnight at 4°C. After washing three times with Tris-buffered saline containing Tween 20 (TBST), the membrane was incubated with second antibody (diluted at 1:2000 and subsequent application of the standard avidin biotinylated peroxidase complex assay) for an additional 1.5 hours at room temperature. Band signals were detected using a chemiluminescence system (Bio-Rad Laboratories Inc, USA) and analyzed using Image LAB software.

Proteins were extracted entirely by utilizing RIPA lysis buffer (Cod. P0013B, Beyotime, Shanghai, China), supplemented with 1% cocktail of phosphatase inhibitor and 1% cocktail of protease inhibitor. Concentrations were quantified using a BCA protein assay kit (Cod. P0010, Beyotime, Shanghai, China). The lysates were combined with 5x SDS-PAGE sample buffer, boiled for 10 min, and then allowed to cool to room temperature. Equal quantities of protein were loaded onto SDS-PAGE gels (Bio-Rad, California, United States), followed by transfer onto PVDF membranes (Sigma-Aldrich, St. Louis, United States) for immunoblot analysis. Membranes were blocked for 1 h at room temperature (RT) in 5% evaporated milk diluted in Tris-buffered saline (TBS) and 0.1% Tween 20 and incubated with the following primary antibodies overnight at 4°C: anti-YAP (1: 1000, Cod. 14074S, Cell Signaling, Danvers, United States), anti-p53 (1: 1000, Cod. sc-126, Santa Cruz, Dallas, United States), anti-p21 (1: 1000, Cod. 2,947, Cell Signaling, Danvers, United States), anti-p16 (1: 1000, Cod. sc-166760, Santa Cruz, Dallas, United States), anti-β-actin (1:1000, Cod. GB15001-100, Servicebio, Wuhan, China), anti-GAPDH (1:1000, Cod. GB15002-100, Servicebio, Wuhan, China). After washing and incubating with the secondary antibodies (1: 10,000, Cod. K1221 and K1223, APExBIO, Huston, United States) for 1 h at RT, immunoreactive proteins were visualized by the ECL (Cod. BL520A, Biosharp, Hefei, China) plus chemiluminescence system (Bio-Rad, California, United States) following the manufacturer’s instructions. Protein bands were quantified using ImageJ software.

Protein extraction from lung tissues and cells was performed as previously described. 20 mg protein samples were separated by 8-12% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Massachusetts, USA). After blocking, the membrane was incubated overnight at 4 °C with specific primary antibodies (anti-β-actin (Affinity, T0022), anti-Nrf2 (Affinity, AF0639), anti-p53 (Proteintech, 60283-2-Ig), anti-NLRP3 (Affinity, DF7438), anti-ASC (Affinity, DF6304), anti-Caspase1 (Affinity, AF5418), anti-Cleaved-Caspase1 (Affinity, AF4005), anti-GSDMD-N (Affinity, AF4012)), followed by incubation with secondary antibodies (Abcam, ab205718, ab6789) at room temperature for 2 h. Protein bands were visualized using a chemiluminescence system (Bio-Rad, USA), with β-actin as a reference.

The total protein from cells, clinical tissues and in vivo tumours was extracted using RIPA lysis buffer that contained 1% phenylmethylsulphonyl fluoride (PMSF). The protein concentrations were then measured using a bicinchoninic acid test kit from Beyotime in Nantong, China. Proteins underwent 10% SDS/PAGE separation, were then transferred to PVDF membranes with a 0.2 µm thickness and were blocked with TBST solution containing 5% non‐fat milk. Following an overnight incubation with primary antibodies (Table S6) at 4°C, the membranes were treated for 1 h at 37°C with secondary antibodies conjugated with horseradish peroxidase at a dilution of 1:5000. A chemiluminescence system (Bio‐Rad, California, USA) and an EasySee kit (TransGen Biotech, Beijing, China) were used to visualise protein bands. Using ImageJ software, the intensities of the bands were measured.