Anti mmp 9

Berberine (purity: ≥98%) and cisplatin (purity: ≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-MMP-2, anti-MMP-9, anti-Bcl-2, anti-Bax, anti-CyclinD1, anti-CDK4, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-P38, and anti-phospho-P38 primary antibodies were purchased from Bioss (Beijing, China). Goat antimouse IgG and goat antirabbit IgG secondary antibodies were purchased from Life Science (Santa Cruz, CA, USA).

The mouse xenograft tumor tissues were homogenized in the RIPA lysis buffer with PMSF, phosphatase and protease inhibitors (Keygen, Nanjing, China). The concentrations of total proteins in each sample were detected by bicinchoninic acid (BCA). Protein samples of each loading was separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) gels and transferred onto polyvinylidene difluoride membrane (Millipore, Danvers, MA, USA). The polyvinylidene difluoride membranes were blotted with anti-KLF4, anti-BMI1, anti-MMP-2, anti-MMP-9, and anti-β-actin (Bioss, London, UK). The immunoblots on the membranes were developed with the enhanced chemiluminescence reagent. The band intensity was determined by densitometric analysis using IMAGEJ software for relative quantification of protein levels by normalized with β-actin in the tumor.

IL-1β was obtained from T&L Biotechnology Co., LTD. (Beijing, China). DMEM medium and FBS were obtained from Genetime Biotechnology Co., Ltd. (Shanghai, China). Primescript™ reverse transcription kit and SYBR^® Premix Ex Taq™ II were purchased from TransGen Biotech (Beijing, China). Antibodies including anti-MMP-1, anti-MMP-3, anti-MMP-9, anti-ADAMS-4, anti-ADAMS-5, anti-collagen, anti-aggrecan, anti-iNOS, anti-COX-2, anti-p-p38, anti-p-ERK1/2, and anti-p-p65 were all obtained from Bioss Biotechnology Co., Ltd. (Beijing, China). GAPDH was purchased from Abcam Inc. (Cambridge, U.K.). The sequences of primers used in the present study was obtained from Tsingke (Beijing, China). ELISA kits were purchased from MSKBIO Technology Ltd. (Wuhan, China).

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).

Digoxin was purchased from Aladdin (London, Ontario, Canada). Doxorubicin was obtained from Dalian Meilun Biological Product Factory (Dalian, Liaoning, China). Cisplatin and verapamil were purchased from Energy Chemical (Shanghai, China). Anti-CyclinD1, anti-Cdc2, anti-CyclinB1, anti-HIF1α, anti-p-Rb (phospho S780), and anti-β-actin antibodies, as well as anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies, were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p21 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-Integrinβ1 (phospho T788+T789) was from Abcam (Cambridge, MA, USA). Anti-MMP2 and anti-MMP9 were obtained from Bioss (Beijing, China). Matrigel and Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis-detection kits were from BD Biosciences (San Jose, CA, USA). Propidium iodide (PI) was from Sigma-Aldrich (St. Louis, MO, USA). Human VEGF-A kit was purchased from Jianglai biotech (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) reagent was from Amresco (Solon, OH, USA).