Af623

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Antibodies against Angpt-2 (AF623) (R&D Systems, Minneapolis, MN), mAngpt-2 (Clone #748246) (R&D Systems, Minneapolis, MN), Tie2 (C-20) (sc-324, Santa Cruz Biotechnology, CA), pTie2 (Y1102/Y1100) (R&D systems, Minneapolis, MN), Gr-1 (AbD Serotec), Lcyopersicon esculentum agglutinin (LEA, tomato lectin) (Vector Laboratories, Burlingame, CA), Akt (11E7) (Cell Signaling Technology, Cambridge, UK), pAkt (Ser 473) (Cell Signaling Technology, Cambridge, UK), pJNK (G-7) (sc-6254, Santa Cruz Biotechnology, CA), JNK (Cell Signaling Technology, Cambridge, UK) and GAPDH (FL-335) (Santa Cruz Biotechnology, CA) were utilized. As secondary antibodies we used goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, CA) and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, CA). The FDA approved drug library was obtained from Enzo Life Sciences (Farmingdale, NY).

The Bm7 and Brmx2 human lung cancer cell line was established as previously described^{14 (link)}. The mouse lung cancer cell line TC-1 was kindly provided by Dr. T.C. Wu^{46 (link)}. A549 cell line was obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). Human umbilical vein endothelial cells (HUVECs) was kindly provided by Dr. Jeng-Jiann Chiu in National Health Research Institute, Taiwan. Human brain microvascular endothelial cells (HBMECs) and primary human astrocytes are purchased from ScienCell Research Laboratory (Carlsbad, CA, USA). Experiments were performed within 6 months of receipt from the cell bank to ensure cells’ ability. All cells used in this study were confirmed to be mycoplasma-free. The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore). The activity of PLAT in different dilutions of the conditioned medium was detected with the AssaySense Human PLAT Immuno-Chromogenic Activity Assay Kit (Assaypro, St. Charles, MO) in 2-day cultured cells.

Human brain microvascular endothelial cells (HBMECs) were seeded onto fibronectin pre-coated 12-well μ-slides (ibidi GmbH, Martinsried, Germany) with a cell density of 8 × 104/mL for overnight culturing. HBMECs were then treated with concentrated conditioned medium from control and knockdown cells for 24 h. To neutralize ANGPT2, the anti-ANGPT2 antibody (10 μg/mL; AF623, R&D) was added to the concentrated conditioned medium for 1 h of incubation, and then this medium was used to treat HBMECs. After culturing, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS (PBS-T) at 4 °C for 10 min. After blocking with 4% BSA in PBS at 37 °C for 30 min, cells were stained with anti-human VE-cadherin antibody (#2158, Cell Signaling) and rhodamine conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). DAPI was used for nucleus staining. Slides were mounted and viewed under a fluorescence microscope (Zeiss Axio Observer Z1) and analyzed using the AxioVision software (Zeiss). The length of VE-cadherin reflected by fluorescence was calculated by the ImageJ program and normalized to the cell number.