E cadherin

Briefly, tissue sections were sequentially treated following the procedures of deparaffinage, rehydration, and antigen retrieval. Endogenous peroxidase was blocked by incubation in 3% H₂O₂ for 10 min at room temperature. The sections were then incubated with anti‐Ki‐67 (Sangon), E‐cadherin (EnoGene), or N‐cadherin (EnoGene) antibodies overnight at 4 °C. Finally, HRP‐conjugated secondary antibody and diaminobenzidine (DAB) solution (Solarbio) were used to detect the signals. Slides were photographed under a microscope. The results were represented by the ratio of positively labeled cells.

MTT, PFT-α, TGF-β1, di-2-pyridylketone, RPMI-1640, LY294002, and other chemicals were purchased from Sigma-Aldrich (Shanghai, China). Fetal bovine serum was purchased from Every Green Zhejiang Tianhang Technology Co. Ltd. (Hangzhou, China). Antibodies of vimentin, slug, snail, and p53 were purchased from Boster (Wuhan, China). Antibodies of AKT, p-AKT, mTOR, p-mTOR, E-cadherin, and Gapdh were purchased from EnoGene (Nanjing, China). 4′-Demethylpodophyllotoxin was purchased from Shanghai PureOne Biotechnology (Shanghai, China).

HepG2 cells were first cultured in a 24-well plate with cover glass overnight. Following Ptox^Dpt treatment for additional 24 h, cells were first fixed with 4% paraformaldehyde in PBS for 15 min at 37°C and then permeabilized with 0.5 % Triton X-100 in PBS for 20 min. After blocking with 3% BSA in PBS for 60 min, the cells were incubated with combined vimentin with E-cadherin (EnoGene) primary antibody based on protocol recommended by the company; at 4°C, the plate was shaken overnight. Next, removing the primary antibodies and washing with PBS, the cells were further incubated with fluorescence-labeled secondary antibody for 3 h at room temperature. After removing the secondary antibody, the cells were further counterstained with DAPI. Finally, a confocal laser scanning microscope (Nikon Eclipse Ts2, Japan) was used to visualize the cells; the representative cells were selected and photographed.