Mmp10

Tumors from mice were stained with IHC for Ki‐67 and MMP10. Briefly, specimens were incubated overnight at 4°C with antibodies specific to Ki‐67 (1:100; Abcam) and MMP10 (1:100; Abcam) in an avidin‐biotin complex method. The signal was later amplified and visualized with a secondary antibody (Beyotime), followed by 3′‐diaminobenzidine chromogen.⁷, ³⁰, ³¹

EC109 and EC9706 cells were seeded onto sterile coverslips (WHB‐24‐CS, diameter: 14 mm) at a density of 5 × 10⁵ cells/coverslip and incubated for 4 h. Cells were then fixed with 4% formaldehyde and coverslips with fixed cells incubated overnight at 4°C with antibodies specific to E‐cadherin (1:100; Abcam), N‐cadherin (1:100; Abcam), Ki‐67 (1:100; Abcam) and MMP10 (1:100; Abcam). Fluorescence‐conjugated secondary antibodies (1:100; Beyotime) were used for 2 h at room temperature. DAPI (Beyotime, China) was used to stain the nuclei.³, ²⁹

The cultured cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor mixtures (Thermo). Equal amounts of proteins were separated by 10% SDS–PAGE and then transferred to nitrocellulose filter membranes (Whatman GmbH, Maidstone, Kent, UK). The membranes were blocked at room temperature for 1 h with 5% non-fat milk and subsequently incubated with primary antibodies directed against AJUBA, ERK1/2, p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), MMP10 and MMP13 (Abcam, Cambridge, UK) at 4°C overnight. β-Actin (Epitomics, Hangzhou, China) was used as a loading control. After washing with TBST, the blots were incubated with the appropriate HRP-conjugated secondary antibody at room temperature for 1 h and then developed using enhanced chemiluminescence (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The signals were quantified using ImageJ software.

Proteins were extracted from samples using radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) according to the manufacture's protocol. After gel electrophoresis, PVDF membranes were blocked in 5% milk/PBS-T for 2 h followed by overnight incubation at 4°C with antibodies against HPSE (Proteintech, Chicago, IL, USA; 1:1000, 66226-1-Ig), phosphor-p38 (Cell Signaling, Beverly, MA, USA; 1:2000, 4511), p38 (Cell Signaling; 1:2000, 8690), MMP1 (Proteintech; 1:1000, 10371-2-AP), PCOLCE (OriGene Tech, Rockville, MD, USA; 0.5 μg/mL, TA337676), MMP10 (Abcam, Cambridge, MA, USA; 1:1000, ab199688), CEACAM6 (Abcam; 1:500, ab78029) and β-actin (Sigma- Aldrich, St Louis, MO, USA; 1:5000, A5316). After washes and incubation with respective horseradish peroxidase-conjugated secondary antibodies for 2 h, protein bands were visualized using the SuperSignal West Pico maximum sensitivity substrate (Pierce, Rockford, IL, USA).