25 cm2 flask

Experiments were carried out on human colon adenocarcinoma cell lines HT-29 and LS180, human colon epithelial cell line CCD 841 CoTr and human skin fibroblasts HSF. HT-29, LS180 and CCD 841 CoTr cell lines were obtained from the European Collection of Cell Cultures (Centre for Applied Microbiology and Research, Salisbury, UK). HSF were obtained with the outgrowth technique from skin explants from young volunteers in the Department of Virology and Immunology, UMCS, Lublin (Poland).
HT-29, LS180, CCD 841 CoTr and HSF cells were cultured in 1:1 mixture of DMEM and Nutrient mixture F-12 Ham. Media were supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL). The medium was changed every two days. The cells were grown in 25 cm² flasks (Nunc, Roskilde, Denmark) and kept in a humidified atmosphere with 5% CO₂ at 37 °C or 33 °C (CCD 841 CoTr).

When 80% confluence in 25 cm² flasks (Nunc, Roskilde, Denmark) was reached, the cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) on ice and then centrifuged at 12000 rpm for 20 min. Supernatants were collected and protein was determined using bicinchoninic acid (BCA) kit (Boster, Wuhan, China). The extracts (20 μg per lane) were fractionated by 10% SDS-PAGE and then transferred onto PVDF membranes (0.45 μm; Millipore, Bedford, MA). After blocking with TBST buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% nonfat milk for 1 h at 25°C, the membranes were incubated with primary antibodies against E-cadherin, N-cadherin (1 : 5000, Epitomics, USA), cytokeratin (1 : 400, Boster, China), EP300 (1 : 1000, Abnova, USA), and β-actin (1 : 5000, CMCTAG, USA) overnight at 4°C. Membranes were washed three times for 5 min with TBST before incubation with horseradish peroxidase-conjugated secondary antibody (1 : 3000, CST, USA) for 60 min at 25°C. The membranes were exposed by chemiluminescence (Millipore, Billerica, MA), and images were acquired by ChemiDoc XRS (Bio-Rad, Hercules, CA). Semiquantification of scanned films was performed using Quantity One-4.6.2 (Bio-Rad).