C myc

Cells were cultured in 24-well plates at a density of 1×10⁵ cells/well, and treated with berberine for 48 hrs. Then, these cells were fixed with PBS containing 2% formaldehyde for 30 mins, and incubated with blocking buffer (Beyotime, Shanghai, China) for 10 mins. After extensive washing, these cells were incubated with primary antibodies overnight against SLC1A5 (Cell Signaling Technology, Danvers, MA, USA) and c-Myc (Proteintech, Wuhan, China). On the following day, these cells were incubated with the secondary antibody, Dylight 488 (Abbkine, Wuhan, China), for two hours, and DAPI for 15 mins. The images of these cells were captured using a fluorescent microscope.

Total protein of cells was extracted with cell lysis buffer (Pierce, Rockford, IL, USA). Cell lysates were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat milk for 2 h and incubated with corresponding antibodies overnight at 4°C. The primary antibodies used were as follows: KRT17 (1:1000, Proteintech, Wuhan, China), active-β-catenin (1:1000, Cell Signaling Technology, MA, USA), c-Myc (1:1000, Proteintech, Wuhan, China), cyclin D1 (1:100, Santa Cruz Biotechnology, CA, USA), MMP7 (1:100, Santa Cruz Biotechnology, CA, USA), GAPDH (1:2000, Santa Cruz Biotechnology, CA, USA), E-cadherin (1:1000, Cell Signaling Technology, MA, USA), Snail (1:500, Proteintech, Wuhan, China), MMP-9 (1:1000, Proteintech, Wuhan, China), and Vimentin (1:2000, Cell Signaling Technology, MA, USA). The membranes were incubated with diluted secondary antibody for 2 h. Protein bands were visualized using an ECL kit (Pierce) and detected using a bioimaging system (DNR, Jerusalem, Israel). GAPDH was used as a loading control. Image J software was used to quantify the results.