Sb 3ct

To inhibit MIF activity, HUVECs were cotreated for 24 h with NS1 and either 100 μM p425 (6,6ʹ-[(3,3-dimethoxy[1,1ʹ-biphenyl]-4,4ʹ-diyl)bis(azo)]bis[4-amino-5-hydroxy-1,3-napthalenedisulphonic acid] tetrasodium salt; Calbiochem, La Jolla, CA, USA) or 50 μM ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid; Calbiochem). In addition, a rabbit anti-MIF polyclonal antibody (10 μg/ml) was used in this study and was purified from recombinant MIF-immunized rabbit serum using a protein G affinity column (GE Healthcare), as previously described [26 (link)]. To inhibit HPA-1, OGT 2115 (Tocris Bioscience, Bristol, UK) was used at the indicated concentration. To inhibit MMP-9, SB-3CT (Abcam, Cambridge, UK) and MMP-9 inhibitor I (Santa Cruz, Dallas, TX, USA) were used at the indicated concentrations. Control mouse and rabbit IgGs were purchased from LeadGene Biomedical (Taiwan).

Cell viability was used to determine the suitable SB-3CT (Abcam, USA) concentration of cell treatment. Briefly, cells were seeded on 96-well plates at a density of 5 × 10⁴ cells per well, and the cells were treated with gradient SB-3CT for 24 h to determine cell viability. Next, cells were treated with 0.5 mg/ml MTT (Sigma, UK) at 37°C for 4 h. The mixture discarded and 100 μl DMSO (Sigma, France) added. After mixing for 5 min, the absorption values were measured at 492 nm on a spectrophotometer (Varioskan Flash, Thermofisher Scientific).

Chemotaxis blockage assays were performed using Costar Transwell inserts after specific chemokine antagonists were added in the supernatants. In brief, the cytokine antagonists used to block the activity of specific growth factors were EYE001 (VEGF antagonist, pegaptanib sodium, Eyetech Pharmaceuticals), etanercept (TNF-α antagonist, Wyeth Pharmaceutical, Japan), ab142180 (MMP-9 inhibitor, Abcam, USA), and SB-3CT (MMP-9/2 inhibitor, Abcam, USA). Each antagonist was dissolved in PBS and added to supernatant of CFs, which were treated with hypoxia for 24 h to a final concentration of 1 μg/mL. Meanwhile, a cocktail of these four antagonists was added in the same final concentration. Supernatant of CFs for hypoxia 0 h was used as control group and added with normal PBS. All groups were incubated for 2 h before the chemotaxis assay was performed to allow interaction of these factors and their antagonists. After a further incubation of 12 h the migration cells number was counted in chemotaxis assay.