Mmp 7

Briefly, after deparaffinization and hydration, the slides were inactivated endogenous peroxidase by using 3% hydrogen peroxide and heat-pretreated in ethylene diamine tetraacetic acid (PH 8.0) by a microwave oven for 5 min. Then, anti-KIF3B (Abcam, USA, ab152976, 37°C, 2h, 1:200) and secondary antibody (37°C, 30 min) were incubated. Sections were stained with Diaminobenzidine (DAB) and counterstained with hematoxylin. PBS was used as negative control. Other antibodies were as follows: β-catenin, CyclinD1, C-myc, p-GSK-3β^ser9, E-cad, Vimentin, MMP-2 and MMP-9 (Bioworld Technology, all at dilution 1:200), Dvl2, MMP-7, Slug and Snail (Bioss, all at dilution 1:200). Independent Histologic and IHC evaluation were conducted by two pathologists. IHC staining of KIF3B was scored as described previously (43 (link), 44 (link)). The scores of staining intensity (0, none; 1, weak; 2, intermediate; and 3, strong) and positive tumor cell proportion (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, > 2/3) were summed up to obtain a total score (ranging from 0 to 8).

Total proteins were extracted from fresh tissues and cultured cells using RIPA lysis buffer containing protease inhibitor PMFS. Nuclear proteins from cells were prepared according to the manufacturer’s instructions of Nuclear Protein Extraction Kit (Solarbio, China). Western blot was performed as described previously (42 (link)). Antibodies used in the study were as follows: KIF3B and Vimentin (Santa Cruz Biotechnology, all at dilution 1:1,000), GAPDH, β-catenin, GSK-3β, p-GSK-3β^ser9, Cyclin D1, C-myc, E-cadherin, MMP-2, MMP-9 (Abcam, all at dilution 1:1,000), Dvl2, MMP-7, Slug and Snail (Bioss, all at dilution 1:1,000), β-actin, Histone H3 (TransGen Biotech, all at dilution 1:4,000).