Angiopoietin-2

Human umbilical vein ECs (HUVECs, ATCC, Manassas) were seeded in confluent conditions (8 × 10⁵ cells/well) in 6-well plates. HUVECs cultured in Human Endothelial Medium (LL-0003, Lifeline Cell Technology, Frederick) for 24 h were pretreated with 100 ng/ml human angiopoietin-1 (ANGPT1, 923-AN/CF, R&D Systems, Minneapolis), 100 ng/ml angiopoietin-2 (ANGPT2, 623-AN/CF, R&D Systems), 30 μg/mL 4E2, or 2.5 μM AKB-9778 for 20 min and were then treated with 100 ng/mL human VEGF-165 protein (293-VE/CF, R&D Systems) for 10 min. When indicated, CHO-K1 cells expressing human Tie2 or mouse Tie2 were maintained in DMEM (Thermo Fisher, Waltham) supplemented with 10% FBS and 200 ng/ml hygromycin (Thermo Fisher).

All serum samples, just after centrifugation at 3000 rpm for 10 min, and all CSF samples were immediately stored at − 80 °C until cytokine analysis, other than IL-6. The serum and CSF IL-6 levels were measured on a single detection immediately after centrifugation at room temperature using the electrochemiluminescence immunoassay according to the manufacturer’s instruction (Roche Diagnostics K.K., Tokyo, Japan). The serum cytokine concentrations relevant to the blood vessel regeneration were measured using the MILLIPLEX® (Merck Millipore, Darmstadt, Germany) human angiogenesis/growth factor magnetic bead panel 1 with a single detection, according to the manufacturer's instruction. Fluorescence intensity from the immunoassay was acquired and analyzed using xPONENT 4.2 Software (Luminex Corporation, Austin, TX, USA). The measured cytokines relevant to the vascular remodeling included epidermal growth factor, angiopoietin 2, granulocyte-colony stimulating factor (G-CSF), bone morphogenetic protein-9 (BMP-9), endoglin, leptin, hepatocyte growth factor (HGF), placental growth factor, vascular endothelial growth factor (VEGF)-C, VEGF-D, fibroblast growth factor-2 (FGF-2), and VEGF-A. Values under the dynamic range were replaced by half of the lower sensitivity limit.

Western Blotting analysis was performed as previously described (25 (link)) in both HGC27-S/R and KATOIII-S/R. Briefly, for each experimental condition the cells were lysed in ice-cold lysis buffer (50 mM Tris, 10 mM EDTA, 1% v/v Triton-X100), supplemented with the protease/phosphatase inhibitor cocktail set (Merck KGaA, Darmstadt, Germany), incubated on ice for 15 minutes and centrifuged at 13,000 × g for 15 min at 4°C. Separate cytoplasmic and nuclear protein fractions were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific Inc., MA USA) following the manufacturing procedures. Protein extracts were quantified by Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc., MA USA) and 40 μg of total protein extract or nuclear/cytoplasmic fraction were subjected to SDS-PAGE and immunoblotted with the following antibodies: P-gp (1:250, rabbit polyclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA), MRP1 (1:500, mouse clone MRPm5, Abcam, Cambridge, UK), BCRP (1:500, mouse clone BXP-21, Santa Cruz Biotechnology Inc.), Phospho-βcatenin (Ser675) and βcatenin (1:1000 Cell Signaling, Beverly, MA, USA), NFkB (1:1000, Santa Cruz Biotechnology Inc.), Phospho-cyclin B1 (Ser147) and cyclin B1 (1:1000 Cell Signaling, Beverly, MA, USA), Beta III Tubulin (TUBβIII 1:2000 Abcam, Cambridge, UK), Phospho-c-Myc (Ser62) and c-Myc (1:1000 Cell Signaling, Beverly, MA, USA), Phospho-SAPK/JNK (Thr183/Tyr185) and JNK2 (1:1000 Cell Signaling, Beverly, MA, USA), Phospho-c-Jun (Ser63) and c-Jun (1:1000 Cell Signaling, Beverly, MA, USA), caspase 3/7 (1:1000 Cell Signaling, Beverly, MA, USA), VEGF Receptor 2 (VEGFR2 1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) VEGF receptor 3 (VEGFR3 1:200, Abcam, Cambridge, UK), Angiopoietin 2 (Ang 2 1:500, R&D Systems, Minneapolis, MN, USA), VEGFA (Abcam, Cambridge, UK), Fibronectin1 (FN1 1:400; Invitrogen), ALIX (1:1000; Abcam, Cambridge, UK), CD81 (1:500; Invitrogen) and GAPDH (1:1000 Abcam, Cambridge, UK). Subsequently, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). An enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) was used. A Chemidoc XRS+ and the Bio-rad software (Bio-Rad, Hercules, CA, USA) was used to observe and analyze the chemiluminescence signals from proteins. Nuclear protein extracts have been normalized using stain free technology, using Image Lab Software (Bio-Rad, Hercules, CA, USA). Total protein expression was quantified using the ImageJ software (http://rsb.info.nih.gov/ij/).