P aminophenylmercuric acetate apma

Antibodies against MMP-9 (ab228402), V5 (ab27671), Integrin β3 (ab119992), ICAM-1 (ab222736), VCAM-1 (ab134047), TRAF-6 (ab33915) and β-actin (ab8227) were purchased from Abcam. Anti- Integrin β5 (#3629), and NF-κB proteins (NF-κB Pathway Sampler Kit #9936) were purchased from Cell Signaling Technology. Pierce™ Protein A/G Plus Agarose was obtained from Invitrogen. LPS, rhodamine B isothiocyanate-dextran, and p-aminophenylmercuric acetate (APMA) were purchased from Sigma-Aldrich. Murine recombinant MMP-9 (R&D, 909-MM), MMP-9 ELISA kits (MMPT90), IL-6 ELISA kits (M6000B), TNF-α ELISA kits (DY410), CXCL-1 ELISA kits (MKC00B), and MPO ELISA kits (DY3667) were from R&D Systems. MMP-9 neutralizing monoclonal antibody (IM09L) was from Sigma-Aldrich. NF-κB inhibitor, PDTC, and ROS scavenger, N-acetyl-L-cysteine (Nac), were obtained from MedChem Express (Shanghai, China). LipoJet™ reagent and GeneMute siRNA transfection reagent were purchased from SignaGen (Ijamsville, MD, USA).

The OPN and MMP-9 plasma concentrations were measured using ELISA kits (R&D Systems Europe, Ltd., Abingdon, UK). The MMP-9 assays recognised both pro- and active forms. Plasma samples were diluted and the immunoassay was performed according to the instructions of the manufacturer. All assays were carried out in triplicate. The minimum detectable dose of OPN and MMP-9 was <0.024 and 0,156 ng/ml, respectively. The optical density was measured at 450 nm using a microplate reader (Thermo Labsystems, Santa Rosa, CA, USA). The activities of MMP-9 were measured using specific Biotrak MMP-9 activity assay kits [Amersham Pharmacia Biotech (UK) Ltd., Little Chalfont, UK] according to the manufacturer's instructions. The appropriate standards were included in each assay. In order to measure the total content of the MMPs, the activation of the pro-form of the MMPs was performed using p-aminophenylmercuric acetate (APMA; Sigma-Aldrich Co. LLC. St. Louis, MO, USA).

EC-1 cells were seeded at a low density in six-well plates and maintained for 24 h in the complete medium. Afterward, they were starved overnight and washed in phosphate-buffered saline (PBS), and the medium was replaced with serum-free RPMI 1640 medium, serum-free medium containing 10 ng/mL recombinant human TGF-β1 (PeproTech, Rocky Hill, NJ, USA), serum-free medium containing 25 µmol/L broad spectrum MMP inhibitor GM6001 (Millipore, Darmstadt, Germany), and serum-free medium containing both TGF-β1 and GM6001. Additionally, to explore the role of MMP-9 in EMT, EC-1 cells were seeded at low density (2×10⁵) in six-well plates for 24 h in the complete medium and starved overnight. Recombinant human MMP-9 protein (R&D Systems, Minneapolis, MN, USA) was activated using p-aminophenylmercuric acetate (APMA; Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 1 mM at 37°C overnight, and the cells treated with this protein were incubated at 37°C for 48 h. Gelatin zymography was used to determine the presence of the active form of MMP-9.