E cad

UNBS5162, amonafide, and 5FU were purchased from the American MedChemExpress, Monmouth Junction, NJ, USA. Further, reference antibodies Bcl-2 (proteintech, Rosemont, IL, USA; Cat# 12789–1-AP), active-Caspase3 (Abcam, Cam-bridge, UK; Cat# ab32042), Bax (proteintech, Cat# 50599–2-Ig), P70/S6K (Abcam, Cat# ab109393), AKT (Abcam, Cat# ab8805), p-AKT (Abcam, Cat# ab38449), mTOR (Abcam, Cat# ab32028), p-mTOR (Abcam, Cat# ab109268), Cyclin D1 (proteintech, Cat# 12363–1-AP), E-cad (Abcam, Cat# ab40772), Snail (proteintech, Cat# 13099–1-AP), N-cad (Abcam, Cat# ab6528), Twist (proteintech, Cat# 11752–1-AP), Slug (proteintech, Cat#) and GAPDH (proteintech, Cat# 60004–1-Ig) were used in this study. Our electrochemiluminescence kit was obtained from proteintech.

After drug administration, the cells were incubated with radioimmunoprecipitation assay lysis buffer on ice for 30 min. The lysate was centrifuged at 12,000 r/min and 4°C for 30 min and the resulting supernatant was collected. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China). Equal amounts of total proteins (30 μg) were mixed with an equal volume of loading buffer and loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The proteins were then transferred onto polyvinylidene fluoride membranes separately. After blocking with 5% powder skim milk for 1 h, the membranes were incubated with primary antibodies against E-cadherin (E-cad), α-smooth muscle actin (α-SMA), fibronectin (FN), and β-catenin (Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Abcam) at room temperature for 1 h. Protein expression was visualized using a chemiluminescence system (Olympus, Tokyo, Japan).

Cells were placed on ice and washed with pre-cold PBS for three times. Lyse the cells with RIPA buffer and discard the cellular debris by high-speed centrifugation. Take 20 μg cell lysate and an equal volume of 2× loading buffer, boil the cell lysate at 95 °C for 5 min. After 10-min centrifugation, 20 μg protein were loaded on 10% SDS-PAGE. Then, transfer the protein to PVDF membrane (MilliporeSigma, USA). After 1 h blocking with 5% defat milk, PVDF membrane was incubated with primary antibodies of mTOR, SYK, E-Cad, Vimentin, Collagen I, α-SMA, and VEGFA (Abcam) prepared with 1× TBST. And incubate in the refrigerator overnight. Next, the PVDF membrane was incubated with HRP-conjugated secondary antibodies (Abcam) after three times washing with 1× TBST. Finally, the membrane was treated with ECL solution and imaged under microscopies. All the primary antibodies were purchased from Cell Signaling Technology and diluted at the ratio of 1:1000.