Ab283684

According to the expression of prognostic genes in the gene signature in the TCGA and ICGC databases, we selected five hub genes (PYCARD, AIM2, IL6, GSDMB, and TIRAP) that were differentially expressed between the cancer and normal tissues for validation using quantitative real-time PCR (RT-qPCR). Informed consent about the tissue sample analysis was obtained from each patient before the initiation of the study, and the study protocol was approved by the Institutional Review Board of the Second Military Medical University (SMMU) Cancer Center. A total of 40 paired normal and cancer tissues were used to validate the different expression level of model-related genes. For detailed experiment procedures, please refer to the previous literature published by our laboratory. The primer sequences used are listed in Table S2 (see Supplementary Data). Immunohistochemical (IHC) staining was performed on Changzheng ccRCC tissue microarray using antibody purchased from the Abcam company (PYCARD, ab283684; AIM22, ab93015; IL6, ab9324; GSDMB, ab235540; TIRAP, ab17218; diluted at 1:100–1:200). The Oncomine database (https://www.oncomine.org/resource/login.html) was utilized to validate the different expression of model-related genes in ccRCC and normal tissue.

As described in our previous study [24 (link)], the whole cell lysate was harvested and subjected to SDS-PAGE and transferred into the nitrocellulose transfer membrane. After incubation with 5% (w/v) milk in PBS/0.05% (v/v) Tween-20 for 1 hour, the membrane was incubated with indicated antibodies overnight at 4°C, subsequently followed by incubation with a horseradish peroxidase secondary antibody (Jackson ImmunoResearch) for 1 hour at room temperature. Proteins were detected using an enhanced chemiluminescence (PerkinElmer). The antibodies used in this study were from Abcam: NLRP3 (ab263899, 1 : 2000 for WB), IL-1β (ab254360, 1 : 1000 for WB), caspase-1 (ab179515, 1 : 1000 for WB), ASC (AB283684,1 : 2000 for WB), GSDMD (ab219800, 1 : 2000 for WB), NF-κB (ab207297, 1 : 2000 for WB), phospho-NF-κB (ab239882, 1 : 2000 for WB), β-actin (ab8226, 1 : 4000 for WB), and α-tubulin (ab7291, 1 : 5000 for WB).

Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime) containing protease inhibitors (Roche, Complete Mini, Basel, Switzerland) and quantified using a bicinchoninic acid kit (Beyotime). Then, 50 µg protein was loaded onto 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). At room temperature, the membranes were blocked with TBST (Beyotime) containing 5% skim milk and then respectively incubated with primary antibodies TGF-β1 (1:1000, ab315254, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab283684, Abcam), cleaved-caspase-1 (1:1000, GTX133447, GeneTex, Irvine, CA, USA), GAPDH (1:1000, ab9485, Abcam) and β-actin (1:1000, ab8227, Abcam) overnight at 4 °C, followed by washing and incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2000, ab205718, Abcam) for 1 h. With GAPDH and β-action as the internal parameters, the protein band was assessed utilizing electrochemiluminescence (Seyotin, Guangzhou, Guangdong, China). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was utilized to analyze Gray value.

The processed OSCC cells and the ground tumors were increased with the lysates including RIPA (Beyotime, China) and protease inhibitors (Beyotime, China). The extracted proteins were quantified through BCA method, mixed with appropriate loading, and heated at 100℃ for denaturation. Then same amount (50 μg) of proteins were added to 10% SDS-PAGE, separated by electrophoresis at constant pressure, and transferred to PVDF membrane (Millipore). Next, the membrane with protein was sealed with 5% skim milk for 2 h, exposed to primary antibodies, including ASC (Abcam, ab283684, 1:1000), IL-1β (Abcam, ab254360, 1:1000), IL-18 (Abcam, ab207324, 1:1000), Pro-IL-18 (Proteintech, 10,663–1-AP, 1:1000), NLRP3 (Abcam, ab263899, 1:1000), IL-6 (Abcam, ab9324, 1 µg/ml), Sox4 (Abcam, ab70598, 1:500), JAK2 (Abcam, ab108596, 1:1000), pJAK2 (Abcam, ab32101, 1:1000), STAT3 (Abcam, ab68153, 1:1000), pSTAT3 (Abcam, ab267373, 1:1000) at 4℃ overnight, and secondary antibodies (Abcam) for 1 h. Finally, western blotting was developed after processing with ECL kit (Thermo scientific), and the brightness of each strip can be controlled by adjusting the exposure time.

Proteins were extracted from the pericontusive cortex (width, 3 mm; depth, 3 mm; length, 5 mm) and quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed to quantify the expression of OX1R, TLR4, NF-p65, NLRP3, ASC, and Caspase-1. Bicinchoninic acid (Cat. no. PA115; Tiangen Biotech, China) was used to measure protein concentrations in the tissue lysates. The primary antibodies used for western blotting included anti-OX1R (1:1000, ab224368, Abcam, UK), anti-TLR4 (1:1000, ab13556, Abcam), anti-p65 (1:1000, D14E12, Cell Signaling Technology, USA), anti-NLRP3 (1:500, ERP23094-1, Abcam), anti-caspase-1 (1:500, ab207802, Abcam), anti-ASC (1:1000, ab283684, Abcam), anti-GAPDH (1:2000, 60004-1-lg, Proteintech, China), and anti-HDAC1 (1:2000, ab109411, Abcam). Subsequently, proteins were electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gels and transferred on to polyvinylidene fluoride membranes. Secondary antibodies (1:5000, Zsgb-Bio, China) conjugated to horseradish peroxidase were incubated with the membranes for 60 min at ambient temperature to facilitate primary antibody binding. After western blotting, the Image Lab Software (version 3.0) was used to evaluate the results.