Anti h3

Proteins were loaded onto 4%–20% gradient protein gels (GenScript, SurePAGE, Cat. No. M00655), 4%–20% Precast Protein Plus Gel (Yeasen, Cat. No. 36256ES10) or 10% SDS-PAGE gels at 150 V for 2 h. A wet transformation was performed at 90 V for 90 min in ice-cold transfer buffer. After that, the membranes were blocked in 5% non-fat milk at room temperature for 1 h on a shaking table. Finally, the blocked membranes were incubated in the corresponding antibodies solutions at room temperature for another 3 h. The following antibodies were used: anti-GFP (Abcam, Cat. No. ab290, 1:10 000 dilution), anti-HA (Sigma-Aldrich, Cat. No. H6533, 1:5000), anti-FLAG (Sigma-Aldrich, Cat. No. A8592, 1:5000), anti-H3 (Proteintech, Cat. No. 17168-1-AP, 1:10 000), and horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody (Abcam, Cat. No. ab6721, 1:10 000). The intensities of blotting signals were quantified using ImageJ software (v.1.50i). Uncropped scans of immunoblotting results are shown in Source data.

Same as the previous experiments, MOVAS were treated with starvation for 12 h. ET-1 inducted MOVAS of the pathological group for 24 h. And then, the corresponding drugs stimulated each group for 48 h. Protein was extracted from lysed cells. When we detected NF-κB, we extracted the nuclear protein from MOVAS. While assaying other factors, the total protein was extracted. The SDS-PAGE gel was prepared with a gel preparation kit (Servicebio, CHN). The samples of each group were separated by SDS gel electrophoresis and then transferred to nitrocellulose membranes, added the primary antibodies overnight after blocking and incubation, and incubated with secondary antibody for 1 h the next day. Protein signals were visualized by ChemiDoc System (BioRad). ImageJ 1.53 software was used to calculate the gray value. The main antibodies used in this study are anti-PI3K (Proteintech, CHN, 1 : 3000), anti-Akt (Proteintech, 1 : 1000), anti-phospho-Akt (Proteintech, 1 : 2000), anti-IκB-α (Cell Signaling Technology, USA, 1 : 1000), anti-NF-κB (Proteintech, 1 : 2000), anti-Bax (CST, 1 : 1000), anti-Bcl-2 (Proteintech, 1 : 1000), anti-GAPDH (Proteintech, 1 : 5000), anti-H3 (Proteintech, 1 : 1000), anti-iNOS (CST, 1 : 1000), anti-TNF-α (CST, 1 : 1000), anti-Rabbit IgG (Servicebio, 1 : 3000), and anti-Mouse IgG (Servicebio, 1 : 3000) [23 (link), 24 (link)].

Total protein was harvested from cells using cell lysis buffer (50 mM Tris-HCl (pH 8.0), 4 M urea, and 1% Triton X-100) supplemented with protease inhibitors. To determine the concentration of cell lysates, the BCA kit (KeyGEN BioTECH, Cat. No: KGP902) was employed. Subsequently, proteins of varying molecular weights were separated through SDS-PAGE and transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% skim milk for 2 h and then incubated with primary antibodies overnight at 4 ℃, followed by secondary antibodies for 1 h. The following antibodies were used: anti-ETS1 (Beyotime, Cat. No: AF6812, 1:1,000), anti-E-Cad (ABclonal, Cat. No: A20798, 1:2,000), anti-Vimentin (Proteintech, Cat. No: 10366-1-AP, 1:2,000), anti-Snail1 (Proteintech, Cat. No: 13099-1-AP, 1:1,000), anti-SIRT3 (Santa Cruz, Cat. No: sc-365,175, 1:200), anti-H3K27cr (PTM BIO, Cat. No: PTM-545RM, 1: 1,000), anti-GAPDH (Proteintech, Cat. No: 60004-1-Ig, 1:10,000), anti-H3 (Proteintech, Cat. No: 17168-1-AP, 1:10,000), anti-rabbit IgG (SAB, Cat. No: L3012, 1:10,000), and anti-mouse IgG (SAB, Cat. No: L3032, 1:10,000). The intensity of bands was scanned and then measured using Image J software.

Proteins in the cells and colonic tissues were extracted using RIPA lysis buffer (Boyotime, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) (Beyotime, China). The BCA kit (Beyotime, China) was used to determine protein concentrations. Western blotting was performed with specific antibodies, including anti-YAP (1:1000, Cell Signaling Technology, 14074), anti-JMJD3 (1:1000, Cell Signaling Technology, 3457), anti-EZH2 (1:1000, Cell Signaling Technology,5246), anti-E-cadherin (1:1000, Cell Signaling Technology, 14472), anti- H3K27me3 (1:1000, Cell Signaling Technology,9733), anti-GAPDH (1:3000, Proteintech, 60004-1-Ig), anti-H3(1:1000, Proteintech, 17168-1-AP), and anti-Occludin (1:1000, Proteintech, 27260-1-AP). Images were visualized using the GeneSys software (Thermo, USA). Gray analysis of the Western blot results was performed using ImageJ software.

Total proteins were isolated from nucleus by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) and quantified using BCA method. Protein sample was analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% seperation gel and 5% spacer gel). The antibodies were anti-TOP1, anti-H3 and anti-rabbit IgG (ProteinTech, China). The dilution ratio for anti-TOP1 was 1:2000. The dilution ratio for anti-H3 was 1:500. The dilution ratio for anti-rabbit IgG was 1:2000. The stripes were visualized by ECL methods (SuperSignal West Pico, Thermo, USA) and detected using Azure C300 biosystems (USA). The grey level values were read by Image J.