Anti mmp2

Cell lysates were extracted with cell lysis buffer (Beyotime, Hangzhou, China) and the protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime). The primary antibodies used were as follows: anti-MMP2, anti-TIMP1, anti-GAPDH (Epitomics, Hangzhou, China); anti-neogenin (Santa Cruz Biotechnology), anti-netrin 4 (R&D Systems); anti-phosphorylated Stat3, anti-phosphorylated Akt, anti-phosphorylated ERK, anti-phosphorylated p38 (Cell Signaling Technology, Danvers, MA, USA).

Western blotting analysis was conducted as previously described [41 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-Slug, anti-Twist, anti-MMP-9, anti-MMP-2, anti-pro caspase-3, and anti-active caspase-3 (Epitomics, Burlingame, USA), anti-HIF-1α and anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin (Cell Signaling Technology, Beverly, MA), and poly (ADP-ribose) polymerase (PARP) (Santa Cruz, CA).

The assay of western blot was performed as previously described [12 (link)]. The primary antibodies were as follows: anti- CTTN (Cat # ab33333, Abcam), and anti- MT1-MMP (Cat #ab3644, Abcam); anti-MMP2 (Cat #97779, Abcam), anti- MMP9 (Cat # ab38898, Abcam), and anti-GAPDH antibody (Cat #2118, CST). The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G antibody (Cat # ab6721, Abcam).

The rabbits and mice were euthanized with an overdose of 3% sodium pentobarbital (160 mg/kg i.p.). The rabbit right femoral arteries and mouse hearts containing advanced plaques were prepared as previously described [18 (link)]. Hematoxylin and eosin (H&E) staining was performed for plaque and lumen areas in rabbits as well as for plaque area in mice. Masson’s trichrome staining was performed to determine the collagen content. Immunohistochemical staining was performed to detect macrophages (for rabbits: anti-RAM-11, 1:1200, Dako, Cat#M0633; for mice: anti-CD68, 1:50, Abcam, Cat#ab955), SMCs (for rabbits: anti-α-actin, 1:2000, Sigma, Cat#A2547; for mice: anti-α-actin, 1:500, Abcam, Cat#ab5694), MMP-2 (for rabbits: anti-MMP-2, 1:400, Bioss, Cat#bs-4605R; for mice: anti-MMP-2, 1:50, Abcam, Cat#ab37150), and MMP-9 (for rabbits: anti-MMP-9, 1:400, Bioss, Cat#bs-4593R; for mice: anti-MMP-9, 1:50, Abcam, Cat#ab38898). Images of the stained sections were obtained using an Olympus IX70 microscope (Olympus, Tokyo, Japan). The amount of collagen, macrophages, SMCs, MMP-2, and MMP-9 were measured using computer-assisted color image analysis software (Image-Pro Plus, version 6.0, Media Cybernetics, Inc., Silver Spring, MA, USA).

Cells were lysed in RIPA Lysis Buffer (MA0151, Dalian Meilun Biotechnology Co., Ltd., China) containing a protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of total protein from different samples were separated by SDS-PAGE gels at 100 V for 1.5 h and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Then, the membranes were blocked with 5% skim milk powder in TBST for 1 h at room temperature and treated with specific primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). Each band was detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Anti-GAPDH, anti-CREB3, anti-Bcl-2, anti-mmp2, anti-Vimentin and anti-E-cadherin antibodies were purchased from Abcam (Cambridge, UK). The anti-c-Jun, anti-cleaved-caspase 3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); anti-mmp9 antibody was purchased from ABclonal (Boston, MA, USA); and anti-N-cadherin antibody was purchased from Proteintech (Chicago, USA). Primary Antibody Dilution Buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).

Celecoxib, cisplatin, 5-fluorouracil (5-FU) and prostaglandin E₂ (PGE₂) were purchased from Sigma-Aldrich. Each compound was prepared as 1 mM stock solution in dimethylsulfoxide (DMSO) for dilution into various concentrations. The following mouse or rabbit monoclonal primary antibodies were used: anti-E-cadherin (Abcam), anti-MMP-2 (Abcam), anti-Vementin (Abcam), anti-c-MYC (Abcam), anti-Axin-2 (Abcam), anti-Cyclin-D1 (Abcam), anti- β-catenin (Cell Signaling Technology), anti-Survivin (Cell Signaling Technology), anti-SOX-2 (Cell Signaling Technology), anti-p-GSK-3β (Cell Signaling Technology), anti-GAPDH (Beyotime Biotechnology) and anti-Tubulin (Beyotime Biotechnology). HRP conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Beyotime Biotechnology.