Anti e cadherin

Tissue sections were prepared by embedding fixed tumor tissue in paraffin, then dewaxing and rehydrating them. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0 or 9.0) at 98 °C for 20 min. Immunohistochemical staining (IHC) was performed on the tissue sections using an automatic tissue processor (Bond-Max; Leica Microsystems, Wetzlar, Germany) according to the standard protocol. Briefly, the sections were incubated with primary antibodies anti-E-cadherin (Proteintech), anti-phospho-Erk1/2 (Biolegend), and anti-cleaved-Caspase 3 (CST). The sections were then treated with HRP-conjugated secondary antibody (BOND Polymer Refine Detection (Leica)) according to the standard protocols of the instrument after washing. The tissue sections were stained with diaminobenzidine and counterstained with hematoxylin. The resulting tissue slides were examined using a BZ-X710 microscope (Keyence, Osaka, Japan).

Total cell extracts were subjected to 12% SDS-PAGE and separated proteins transferred to polyvinylidene fluoride membrane. Western blot was performed using standard protocols described previously [46 (link)]. Rabbit anti-DEK, anti-E-cadherin, anti-vimentin, anti-MMP2, and anti-MMP9 antibodies were purchased from Proteintech (Chicago, IL, USA). Mouse anti-GAPDH antibody was obtained from Chemicon (Temecula, CA, USA).

The cells’ protein was collected by RIPA lysis buffer (Byotime, P0045), then the BCA protein assay kit (Biosharp, BL521A) normalizes the samples. The cell extracts were boiled with Sample Loading Buffer (Biosharp, BL529A) for 5 min. After separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, Germany), the membranes were blocked in 5%BSA and incubated with primary antibodies at 4°C overnight. The primary antibodies are listed as follows: anti-PLAU (1:2,000, Proteintech, 17968-1-AP), anti-E-Cadherin (1:5,000, Proteintech, 20874-1-AP), anti-N-Cadherin (1:2,000, Proteintech, 22018-1-AP), anti-TWIST1 (1:1,000, Proteintech, 25465-1-AP), anti-SNAI1 (1:1,000, Proteintech, 13099-1-AP). Then the membranes were incubated with secondary antibody (Biosharp, BL003A) and the proteins were visualized by ECL enhanced chemiluminescence substrate (Millipore).

SDS‐PAGE and western blot were conducted using the following antibodies: anti‐GAPDH (BM3874, Boster), anti‐TRIM55 (A15917, Abclonal), anti‐MMP2 (orb12416, Biorbyt), anti‐MMP9 (27306‐1‐AP, Proteintech), anti‐N‐Cadherin (22018‐1‐AP, Proteintech), anti‐Bak (A0498, Abclonal), anti‐Bax (sc‐7480), anti‐Bcl2 (ET7110‐51, HuaBio), anti‐Caspase 3 (ET1602‐39, HuaBio), anti‐c‐Myc (AF0358, Affinity), anti‐p21/Cip1 (27296‐1‐AP, Proteintech), anti‐E‐Cadherin (20874‐1‐AP, Proteintech), anti‐P27/Kip1 (NBP1‐32213, Novus), anti‐survivin (NBP2‐48494, Novus), mouse anti‐HA‐Tag mAb (AE065, Abclonal), and mouse anti His‐Tag mAb (AE003, Abclonal). Protein bands were developed with a chemiluminescence kit (ThermoFisher).

Western blot assay was performed as the previous study described [48 (link)]. Total proteins were extracted using RIPA buffer (#FD011, Hangzhou Fude Biological Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). In total, 1 μg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). After blocked with 5% Non-Fat Milk and incubated with specific antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation, the membrane was imaged with imager (Biorad ChemiDoc MP, Biorad). The antibodies used in this study were listed: anti-GAPDH (#5714, Cell Signaling Technology), anti-METTL1 (#ab157097, Abcam), anti-E-cadherin (#20874-1-AP, Proteintech), anti-N-cadherin (#66219-1-AP, Proteintech), anti-VIMENTIN (#10366-1-AP, Proteintech), anti-MMP2 (#10373-2-AP, Proteintech), anti-CDK4 (#11026-1-AP, Proteintech), anti-P16 (#18769, Cell Signaling Technology), anti-ATF3 (#ab254268, Abcam).