Anti β actin

The frozen samples were lysed with RIPA buffer. Their protein concentrations were identified using the bicinchoninic acid assay method. The proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% gel and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% (w/v) bovine serum albumin in Tris‐buffered saline with Tween 20 for 1 h. These membranes were incubated using P‐Smad2 antibody (1:1000, Affinity), anti‐P‐Smad3 antibody (1:800, Affinity), anti‐Atg7 antibody (1:1200, Affinity), anti‐LC3II /I antibody (1:800, Affinity), anti‐ACTA antibody (1:800, Santa Cruz), and anti‐β‐actin (1:1000, Affinity) and were diluted in Tris‐buffered saline with Tween 20 buffer overnight at 4°C. The immunoblots were then incubated with peroxidase‐conjugated secondary antibody (1:3000; ZB2305, ZSGB‐BIO, Beijing, China) for 90 min at room temperature. Band intensities obtained by western blot assay were determined using the ImageJ open‐source software (National Institutes of Health, Bethesda, MD, USA).

Proteins were extracted from cell lysates in lysis buffer and used to quantify protein levels. Proteins were separated on polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoprobed with specific antibodies. The proteins were detected using a chemiluminescence reagent kit purchased from Thermo Fisher Scientific. The imager station captured the images (Odyssey Software Version 5.2, LI-COR Biosciences). Anti-TRIO (Affinity, DF2685), anti-TRIOBP (Proteintech, 16124-1-AP), anti-β-actin (Affinity, AF7018), and anti-GAPDH (Affinity, AF7021) antibodies were purchased from Affinity Biosciences LTD. Anti-α-SMA antibodies were purchased from Abcam (Abcam, ab124964). Anti-vimentin (Proteintech, 10366-1-AP), anti-E-cadherin (Cell Signaling Technology, 14472S), anti-type I collagen (Proteintech, 14695-1-AP) and anti-fibronectin (Proteintech, 15613-1-AP) antibodies were purchased from Proteintech Group.

After different treatments, the BV-2 cells were washed with cold PBS twice, and the cells were fully lysed with RIPA lysate containing 1% protease inhibitor. The lysate was collected and centrifuged at 4 °C at 12,000 rpm for 25 min. Part of the supernatant was taken for protein-concentration determination, and the remaining supernatant was mixed with the loading buffer and boiled at 100 °C for 10 min. Fifty μg of protein were injected into the pore of 12% and 8% SDS polyacrylamide gel to isolate proteins with different molecular weights. The protein was then transferred at a constant voltage to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was immersed in TBST solution containing 5% skim milk and slowly shaken at room temperature for 1 h. Wash the protein band with TBST three times. Anti-β-actin (1:1000) (Affinity, Changzhou, China), Anti-COX2 (1:1000) (Bioss, Beijing, China), anti-NLRP3 (1:1000) (GeneTex, Southern California, CA, USA), and anti-Caspase-1 (1:500) (Wanleibio, Shenyang, China) antibodies were used to incubate the protein bands at 4 °C overnight. On the second day, protein bands were incubated with either goat anti-rabbit (1:10,000) or goat anti-mouse secondary antibody (1:10,000) (ZSGB BIO, Beijing, China) for 1 h. All protein bands were measured by ECL Western blot assay.

Total protein from colonic tissue specimens was isolated and resolved by SDS-PAGE. After transfer onto PVDF membranes, 5% skimmed milk was utilized for blocking. The blocked membranes underwent incubation with anti-TLR4 (AF7017, 1:1000), anti-NF-κB (AF5006, 1:1000), anti-p-NF-κB (AF2006, 1:1000), anti-PI3K (AF6241, 1:1000), anti-p-PI3K (AF3241, 1:1000), anti-Akt (AF6261, 1:500), anti-p-Akt (AF0016, 1:500), anti-IL-1β (AF5103, 1:1000) and anti-β-actin (AF5332, 1:1000) primary antibodies from Affinity Bioscience (Cincinnati, OH, USA) overnight, respectively. Then, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (E-AB-1003; Elabscience, Wuhan, China). Signals were detected with an ECL imaging system (LAS4000, GE, Pittsburgh, PA, USA). ImageJ (National Institutes of Health, Bethesda, Maryland, USA) was used for quantitation.

The total protein content of cells was extracted using lysis buffer (Beyotime Institute of Biotechnology), phenylmethylsulfonyl fluoride solution (Beyotime Institute of Biotechnology), and a protease inhibitor cocktail (bimake). The protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). The 0.45 µm membrane was blocked with 5% skim milk. The membrane was incubated with anti-UBE2S (Proteintech), anti-BOP1 (Proteintech), anti-CDC20 (Proteintech), and anti-β-ACTIN (Affinity Biosciences). The membranes were washed three times and incubated with a secondary antibody (Thermo Fisher Scientific).

Human PCa cell lines (PC3 and C4-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Cat# 10270-106, Gibco, UK) and penicillin–streptomycin combination solution (10 kU/mL penicillin and 10 mg/mL streptomycin, Cat# PB180120, Procell, Wuhan, China) at 37 °C and 5% CO₂. PCa cells were transfected with small interfering RNAs (siRNAs) (General biological system, Anhui, China) using Lipofectamine 3000 reagent (Lot# 2307436, Invitrogen, USA). The sequences of si-cyclin-dependent kinase inhibitor 2C (si-CDKN2C), si-Rac GTPase-activating protein 1 (si-RACGAP1), and the nontargeting control (NC) are shown in Additional file 1: Table S1. The antibodies used were as follows: anti-CDKN2C (Cat# ab192239, Abcam, Cambridge, UK), anti-RACGAP1 (Cat# ab134972, Abcam, Cambridge, UK), anti-β‐actin (Cat# AF7018; Affinity, OH, USA), and anti-β‐tubulin (Cat# AF7011, Affinity, OH, USA). The secondary antibodies included goat anti‐rabbit for CDKN2C, RACGAP1, and β-actin (Cat# AS014, ABclonal, Wuhan, China) and goat anti-mouse for β-tubulin (Cat# S0002, Affinity, OH, USA).

NaHS was obtained from Sigma Chemical (St. Louis, USA); [γ-³²P] ATP was obtained from China Tongfu Co., Ltd (Beijing, China); PKG1, ATP, DT-2, and ACh were obtained from Sigma-Aldrich (St. Louis, MO, USA); LDH test kit was obtained from Nanjing Jiancheng Biotechnology (Nanjing, China); NSE Assay Kit was obtained from Jiangsu Meimian Industrial, Co., Ltd (Jiangsu); Fluo-8 AM, anti-ROCK₂ (catalog number ab125025), anti-Phospho-RhoA Ser188 (catalog number ab41435), and anti-RhoA (catalog number 187027) were purchased from Abcam (San Francisco, California, USA); and anti-β-actin (catalog number AF7018) and goat anti-rabbit IgG secondary antibody (catalog number S0001) were purchased from Affinity Biosciences (Changzhou, China).