Histone h3

The total protein from tissues and cultured HPASMCs were isolated using cell lysis buffer for Western and IP kit (Beyotime, China) and the nuclear protein and cytoplasmic protein were extracted using nuclear and cytoplasmic protein extraction kit (Beyotime) according to the manufacturer’s protocols. Protein concentrations were determined using the BCA protein assay kit (Beyotime). Proteins were loaded and separated in SDS-PAGE (8, 10 and 15%) and immunoblotted with antibodies including HSP110 (Affinity, USA), LC3 (Abclonal, China), Beclin1 (Abclonal), ATG5 (Abclonal), ATG7 (Abclonal), p62 (Abclonal), p-YAP (Affinity), YAP (Santa cruz, USA), p-TAZ (Affinity), TAZ (Proteintech, China), TEAD4 (Abclonal), β-actin (Santa cruz) and histone H3 (Abgent, USA) overnight at 4 °C. Signal intensities of protein band were developed using ECL chemiluminescence kit (Beyotime) and the band densities were measured using gel imaging analysis system (LiuYi, China). The relative protein levels were calculated after normalization with β-action and compared to the appropriate control group.

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).