Mmp 9 inhibitor 1

A rabbit polyclonal anti-mouse DSP (M-300 # sc-33587) antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, United States). Anti-MMP9 (AF909) and Anti-MMP2 (AF1488) antibodies were purchased from R&D SYSTEMS (United States). A monoclonal anti-FURIN (ab183495) and a polyclonal anti-MMP9 (ab38898) antibodies were purchased from Abcam Corp. (Cambridge, United Kingdom). A mouse monoclonal anti-MYC-tag (60003-2-Ig), an anti-beta Actin (60008-1-Ig) and a monoclonal anti-GAPDH (60004-1-Ig) antibodies were purchased from Proteintech (United States). MMP9 Inhibitor I (444278) was purchased from EMD Millipore Corp. (Billerica, MA, United States). MMP2 Inhibitor II (444286) was purchased from EMD Chemicals Inc. (San Diego, CA, United States). MMPs broad-spectrum inhibitor used in the study was obtained from Thermo Fisher Scientific. FURIN Inhibitor I (Decanoyl-RVKR-CMK) (34493) was purchased from Merck Millipore (Darmstadt, Germany). IFKine^TM Red Donkey Anti-Rabbit IgG (A24421) and IFKine^TM Green Donkey Anti-Goat IgG (A24231) were purchased from Abbkine Scientific Co. (California, CA, United States). EnzChek^TM Gelatinase/Collagenase Assay Kit (E12055) was purchased from Thermo Fisher Scientific.

Leukemia cells were labelled with cell trace violet dye (ThermoFisher Scientific) and cultured on ~ 80% confluent meningeal cells. Anti-human CD99 monoclonal antibody clone 0662 (MABF927, EMD Millipore) or isotype control (mouse IgG3, kappa monoclonal; Abcam ab18394) were added at the same time as co-culture. For some experiments, MMP-9 inhibitor I 1 μM (EMD Millipore CAS 1177749-58-4), AP-1 inhibitor T-5224 500 nM (Fisher Scientific) or Batimastat 500 nM (BioVision) were added as well. After 24 h, the supernatant was collected and count beads were used with flow cytometry to determine the number of non-adherent, viable leukemia cells. Labeling of the leukemia cells with cell trace violet facilitated gating out any non-adherent meningeal cells. The ratio of leukemia cells in each condition to cells plated were used to calculate the percentage of non-adherent leukemia cells. For experiments testing the effect of CD99 antibody pre-treatment, Jurkat and human meningeal cells were separately incubated ± CD99 antibody clone 0662 (5 μg/mL) for 24 h, washed to remove excess antibody, combined in a co-culture adhesion assay, and adhesion measured after an additional 4 h.

VSMC were grown in 12- or 24-well plates to ~90 % confluency (day 0). Calcification was induced by a calcification medium (CM) consisting of standard culture medium supplemented with NaH₂PO₄ and CaCl₂ to final concentrations of PO₄³⁻ (2.8 vs. 1.0 mmol/l) and Ca²⁺ (2.7 vs. 1.8 mmol/l), respectively. Cells were treated for up to 9 days as indicated and media were replaced every 2–3 days.
To study their effects on VSMC calcification, the recombinant MMPs-2 and −9 (3 nM; BioTez, Berlin, Germany) and the specific inhibitors for MMP-2 (MMP-2 inhibitor I; 10 μM; Merck Schwalbach, Germany), MMP-9 (MMP-9 inhibitor I; 1 μM; Merck) and both gelatinases (Ro28-2653; 1 μM; Roche Diagnostics GmbH, Penzberg, Germany) were added to CM.

Monoclonal mouse anti‐human ICAM‐1 (clone R6.5) was from ATCC (Manassas, VA) and phycoerythrin‐labeled monoclonal mouse anti‐human ICAM‐1 (clone LB‐2) was from Santa Cruz Biotechnology (Dallas, TX). Alexa Fluor 350‐labeled goat anti‐mouse IgG was from Invitrogen (Carlsbad, CA). Nonspecific mouse IgG was from Jackson ImmunoResearch (West Grove, PA). Green Fluoresbrite® polystyrene particles (100 nm in diameter) were from Polysciences (Warrington, PA). Porous transwell inserts (1.0 µm‐pore size) were from Thermo Fisher Scientific (Waltham, MA). Human sICAM‐1 ELISA kits were from Invitrogen (Carlsbad, CA). ¹²⁵Iodine (¹²⁵I) and Iodogen pre‐coated tubes were from PerkinElmer (Waltham, MA) and Thermo Fisher Scientific (Waltham, MA), respectively. MMP‐9 Inhibitor I and MMP‐2 Inhibitor I were from EMD Millipore (Billerica, MA). Unless specified, all other reagents were from Sigma‐Aldrich (St. Louis, MO).