Anti p65

Co-IP assays were performed using a BeaverBeads™ Protein A/G Assay kit (Beaver, China). NR8383 cells (1 × 10⁶) were lysed in 300 μL IP binding buffer with 1 mM PMSF protease inhibitor on ice for 30 min. Lysates were centrifuged at 17,000 g for 10 min at 4 °C. One-quarter of the supernatant was held as input, and the remaining supernatant was used for the Co-IP assay. Protein A/G agarose beads (50 μL) washed with 200 µL binding buffer were added to 200 μL of lysate and incubated with anti-IgG (1:500, Cell Signaling Technology, Inc.) or anti-H3K27me3 (1:250, Abcam) overnight at 4 °C with slow shaking. The sample was washed with 200 µL washing buffer, and beads were magnetically separated. The immobilized protein complex was eluted at 95 °C in 5X SDS-PAGE Loading Buffer (20 μL) for 10 min. The supernatant was removed, and Western blotting was performed using anti-H3K27me3 (1:1000, Abcam), anti-EZH2 (1:2000, Proteintech) and anti-P65 (1:2000, Proteintech). IgG was used as a negative control.

The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.

CRC cells or liver metastatic tissues were lysed with RIPA Lysis Buffer (Beyotime, China) containing 1% PMSF (Beyotime). Besides, the nuclear protein was isolated by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein from different conditions were separated by 6–14% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk or 1% BSA, the membranes were incubated with following primary antibodies at 4 °C overnight: anti-E-cadherin (1:1000, 60,335–1-Ig, Proteintech, China), anti-N-cadherin (1:1000, 66,219–1-Ig, Proteintech, China), anti-fibronectin (1:500, 15,613–1-AP, Proteintech, China), anti-p-IκBα (1:500, bs-2513R, Bioss, China), anti-IκBα (1:500, bs-1287R, Bioss, China), anti-p65 (1:500, 10,745–1-AP, Proteintech, China), anti-β-actin (1:500, KGAA001, KeyGen, China) and Histone H3 (1:2000, AM8433, ABGENT, USA). After rinsed with TSBT, the membranes were incubated with their corresponding secondary antibodies at 37 °C for 45 min. The protein bands were visualized using the ECL reagent (Beyotime).

EC cells protein were blotted as described in the literature [35 (link)]. In brief, protein was separated by electrophoresis on SDS-polyacrylamide electrophoresis (PAGE) gels, transferred to polyvinylidene difluoride (PVDF) membranes and blocked with blocking solution at room temperature for 1 h. The PVDF membranes were incubated with primary antibody (anti-IDO1, 1:750, Abcam, Cambridge, UK; anti-CXCL10, 1:1000, Saier, Tianjin, China; anti-P65, 1:2000, Proteintech, Wuhan, China; anti-p-P65, 1:1000, Abcam; P105, 1:1000, Abcam; anti-p-P105, 1:1000, Abcam; anti-GAPDH, 1:3000, Bioworld) at 4 °C overnight, then cleaned with 1×PBST buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG/Goat Anti-Mouse, 1:3000, Beyotime, Shanghai, China) for 1 h at room temperature. After the membranes were washed in PBST, the blots were visualized by enhanced chemiluminescence ECL kit (Thermo Fisher Scientific).

Alantolactone (HY-N0038) was obtained from MedChem Express (MCE). Recombinant mouse IL-1β cytokine was provided from R&D Systems (501-RL-010, United States). Solarbio supplied safranin O liquor (Beijing, China). Primary antibodies applied in this research were: anti-iNOS, anti-MMP-13 which were acquired from Abcam (Shanghai, China), anti-COX2, anti-LC3Ⅱ/Ⅰ, anti-P-STAT3, anti-P-P65, anti-P-IκB, anti-P/T-mTOR which were purchased from CST (Beverly, MA, United States), anti-STAT3, anti-MMP-1, anti-MMP-3, anti-ATG5, anti-P62, anti-P65, anti-IκB, anti-P/T-PI3K, anti-P/T-AKT, anti-GAPDH which were afforded from Proteintech Group (Wuhan, Hubei, China) and anti-ADAMTS5 which was supplied from Boster Biological Technology (Wuhan, Hubei, China). Secondary antibodies, Cy3 and FITC Conjugated AffiniPure Goat Anti-Rabbit IgG, DAPI staining solution, collagenase type II and trypsin were got from Boster Biological Technology (Wuhan, Hubei, China). HanBio Inc. (Shanghai, China) served the mRFP-GFP-LC3 adenoviral vectors.

The Magna ChIP^TM HiSens Chromatin Immunoprecipitation Kit (Cat. No. 0025; Millipore) was used to perform the ChIP assay according to the manufacturer's recommendations. Briefly, ≈2 × 10⁶ primary CFs cultured in a 10 cm petri dish were used per immunoprecipitation sample. CFs were fixed for 10 min at room temperature (23–27 °C) and sonicated on a Bioruptor UCD‐200TM‐EX for 16 cycles of 30 s ON and 30 s OFF at high power. DNA bound to NKRF, p65, and p50 was precipitated using anti‐NKRF (Cat. No. 14693‐1‐AP; Proteintech), anti‐p65 (Cat. No. ET1603‐12; HUABIO, Hangzhou, China), and anti‐p50 (Cat. No. 14220‐1‐AP; Proteintech). Rabbit IgG (Cat. No. 2729; Cell Signaling Technology) was used as a control. The precipitated DNA was subjected to PCR following agarose gel electrophoresis and RT‐PCR to calculate the fold enrichment of ChIP DNA using specific HuR promoter primers. These primer sequences were listed in Table S2 (Supporting Information).