N cadherin

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor cocktail. BCA protein quantification kit (Vazame, China) was used to detect the protein concentration. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking with 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies, followed by incubation with HRP-labeled secondary antibody for 1 h at room temperature. Primary antibodies were purchased from Cell Signaling Technology (E-cadherin, 1:1000, #3195; N-Cadherin, 1:1000, #13,116; Snail, 1:1000, #3879; MMP-2, 1:1000, #40,994; MMP-9, 1:1000, #13,667; Phospho-Akt, 1:1000, #4060; Akt, 1:1000, #4691; PI3K, 1:1000, #3011), Bioss (Phospho-PI3KCA, 1:1000, bs-5570R), Proteintech (TEAD4, 1:2000, 12,418–1-AP; Vimentin, 1:1000, 10,366–1-AP), and ImmunoWay (GAPDH, 1:10,000, YM3209). Secondary antibodies were purchased from Elabscience (Goat Anti-Rabbit IgG (H + L), 1:10,000, E-AB-1003; Goat Anti-Mouse IgG(H + L), 1:10,000, E-AB-1001).

The antibodies were bought from Cell Signaling Technology (c‐Myc, CDK4, CDK6, CCND1, BCL2, C‐PARP, PARP, and C‐Caspase 3), Proteintech Group (N‐cadherin, E‐cadherin, MMP9, Vimentin, FBXW7, α‐Tubulin, and HA‐tag), Bioss (c‐Myc for IHC), Abcam (anti‐BrdU), and Beyotime (HRP goat‐rabbit and goat anti‐mouse). Phenylmethylsulfonyl Fluoride (PMSF), RIPA lysis buffer, and BCA protein assay kit were bought from Beyotime. 5‐Fu and DOX were obtained from Med Chem Express and Selleck Chemicals, respectively. 5‐Bromo‐2‐deoxyuridine (BrdU), 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and MG132 were purchased from Sigma‐Aldrich. 4’, ‐6‐Diamidino‐2‐phenylindole (DAPI) and puromycin were obtained from Life Technologies. The Hieff Trans Liposomal Transfection Reagent was purchased from Yeasen. ECL solution was offered by US Everbright Inc.

Protein extraction was executed utilizing RIPA (P0013C; Beyotime) and protein concentration was examined with NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). An equal amount of proteins was separated by 10% sodium dodecyl sulfonate-polyacrylamide gel (P1200; Solarbio) electrophoresis and subsequently blotted onto polyvinylidene difluoride membranes (ISEQ00010; Millipore, Billerica, MA, USA). Thereafter, the membranes were blocked in skim milk for 1 h and cultivated with primary antibodies overnight at 4 °C followed by incubation with secondary antibody (bs-40296G-HRP; Bioss, Beijing, China) at indoor temperature for 1 h. The signal of the bands was visualized by the ECL kit (E411-04; Vazyme, Nanjing, China). The primary antibodies used in this study included Ki67 (bs-23103R; Bioss), Twist1 (bs-2441R; Bioss), E-cadherin (bs-1519R; Bioss), N-cadherin (bs20623R; Bioss) KPNA4 (bs-16804R; Bioss), CyclinD1 (bs-0623R; Bioss), Bcl-2 (bs-33411R; Bioss), Bax (bs-0127R; Bioss) and GAPDH (bs-10900R; Bioss).