MGH7 and H2170 squamous lung carcinoma cell lines were cultured in 5% CO2 at 37 °C in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% (v/v) Fetal Calf Serum (FCS) as previously described41 (link). Bevacizumab (Avastin®) was kindly provided by Roche/Genetech, Indianapolis, USA. VEGFR2 kinase inhibitor KI8751 (cat#676484) was from Calbiochem. SU5416 (cat#S8442) and the inhibitor of Notch signaling FLI-06 (cat#SM0975) were purchased from Sigma-Aldrich. The human recombinant VEGF-A ligands rhVEGF165 (cat#293-VE) and rhVEGF121 (cat#4644-VS) were supplied by R&D Systems, whereas rhVEGF189 (cat#CRV114A) was from Cell Sciences (Canton, USA). The plasmids used in this study were: pcDNA3.1, pcDNA3.1-VEGF165 (kindly provided by Pr David Bates, University of Notthingham, UK). Transfections of plasmid DNA were performed using X-tremeGENE 9 (Roche), according to the manufacturer’s instructions. Cells were analyzed 48 h after transfection. All methods were performed in accordance with the relevant guidelines and regulations.
Rhvegf165
Manufactured by R&D Systems
Sourced in United States
RhVEGF165 is a recombinant human Vascular Endothelial Growth Factor 165. It is a heparin-binding glycoprotein that is a key regulator of angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Egm 2mv, by Lonza (3 mentions)
Alpha minimal essential media alpha mem, by Thermo Fisher Scientific (2 mentions)
Antibiotic and antimycotic solution, by Thermo Fisher Scientific (2 mentions)
Endothelial cell growth medium 2 egm2 mv, by Lonza (2 mentions)
Egm2 mv, by R&D Systems (2 mentions)
Bevacizumab, by Genentech (2 mentions)
Y 27632, by Merck Group (2 mentions)
Fli 06, by Merck Group (1 mentions)
Rhvegf121, by R&D Systems (1 mentions)
Su5416, by Merck Group (1 mentions)
X tremegene 9, by Roche (1 mentions)
Bevacizumab avastin, by Roche (1 mentions)
Rhbmp 2, by R&D Systems (1 mentions)
Wistar rat, by Charles River Laboratories (1 mentions)
T75 culture flask, by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
α mem, by Merck Group (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
12 protocols using rhvegf165
1
VEGFR2 and Notch Signaling Inhibitors
2
Isolation of Rat Bone Marrow-Derived Mesenchymal Stem Cells
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Bone marrow-derived mesenchymal stem cells (rBM-MSCs) were isolated from 8-weeks old male Wistar rats (Charles River Laboratories, Écully, France). Briefly, the animals were anesthetized with a medetomidine/ketamine cocktail and sacrificed by cervical dislocation. Their femora were excised, the epiphyses were removed and the bone marrow was flushed out of the diaphysis with α-MEM (Sigma-Aldrich), passing them several times through a 21 G needle. Erythrocytes were removed by incubating the sample for 10 min in red blood cell lysing buffer (Sigma-Aldrich). The remaining cells were expanded as monolayers in T-75 culture flasks (Nunclon, Nunc, Roskilde, Denmark) in α-MEM supplemented with 10% fetal bovine serum (FBS), 2.5 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1.25 µg/mL amphotericin, with a change of medium at day three to remove the non-adherent cells. The cells used for the in vitro assays were at passage 3–4.
The rhBMP-2 and rhVEGF-165 produced in CHO and SF21 cells, respectively, were purchased from R&D Systems (Minneapolis, MN, USA) and handled according to the manufacturer’s instructions.
The rhBMP-2 and rhVEGF-165 produced in CHO and SF21 cells, respectively, were purchased from R&D Systems (Minneapolis, MN, USA) and handled according to the manufacturer’s instructions.
3
DPSC Capillary Sprouting Assay
4
Differentiation Potential of Dental and Bone Stem Cells
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Human BMSCs derived from iliac crests (Lonza, Basel, Switzerland) and DPSCs isolated from adult third molars (Lonza) were cultured in growth medium (GM) comprising Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, MO, USA). Green fluorescent protein- (GFP-) labelled DPSCs were used to track their localization inside cell constructs. DPSCs at passages 3–5 were transduced with GFP using a lentivirus vector (Sigma-Aldrich) and selected by 1 μg/mL puromycin (Sigma-Aldrich) for at least 1 week.
To induce the endothelial differentiation, each cell type and cell constructs were treated with an endothelial differentiation medium (EM; EGM2-MV (Lonza) supplemented with 50 ng/mL rhVEGF165 (R&D Systems, MN, USA)). For osteogenic differentiation, cell constructs were maintained in osteogenic differentiation medium (OM) comprising GM supplemented with dexamethasone (1 × 10−6 mol/L; Sigma-Aldrich), β-glycerophosphate disodium salt hydrate (1 × 10−2 mol/L; Sigma-Aldrich), ascorbic acid (50 μg/mL; Sigma-Aldrich), and calcium chloride (1 × 10−2 mol/L; Wako). Cells and cell constructs were incubated in a humidified incubator at 37°C with 5% CO2.
To induce the endothelial differentiation, each cell type and cell constructs were treated with an endothelial differentiation medium (EM; EGM2-MV (Lonza) supplemented with 50 ng/mL rhVEGF165 (R&D Systems, MN, USA)). For osteogenic differentiation, cell constructs were maintained in osteogenic differentiation medium (OM) comprising GM supplemented with dexamethasone (1 × 10−6 mol/L; Sigma-Aldrich), β-glycerophosphate disodium salt hydrate (1 × 10−2 mol/L; Sigma-Aldrich), ascorbic acid (50 μg/mL; Sigma-Aldrich), and calcium chloride (1 × 10−2 mol/L; Wako). Cells and cell constructs were incubated in a humidified incubator at 37°C with 5% CO2.
5
Cytotoxicity Evaluation of CS2164 in HUVEC and NIH3T3 cells
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HUVEC and PDGFRβ overexpressed NIH3T3 cells were seeded in 96‐well plates at 5 × 103 cells/well. After 6 h culture for attachment, the cells were cultured in FBS‐free DMEM overnight. HUVEC and PDGFRβ overexpressed NIH3T3 cells were, respectively, treated with recombinant human VEGF 165 (rhVEGF165; R&D Systems, Minneapolis, MN, USA) and recombinant human PDGF‐BB (rhPDGF‐BB; PeproTech, Rocky Hill, NJ, USA) at 100 ng/mL together with CS2164 (or sunitinib as a reference drug) at indicated concentrations. Growth inhibition (GI50) that reduces the activity of mitochondrial aldehyde dehydrogenases by 50% compared to control at 72 h was calculated by MTS (Promega, Madison, WI, USA) test, as described previously.28
6
Vascular Differentiation of Dental Stem Cells
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DPSC (Lonza, Walkersville, MD, USA) and SHED (kindly provided by Songtao Shi) were cultured in Alpha-Minimal Essential Media (Alpha-MEM; Invitrogen, Carlsbad, California) supplemented with 20% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA), 1% Antimycotic and Antibiotic Solution (Gibco, Grand Island, NY, USA) at 37°C and 5% CO₂. Primary Human Dermal Microvascular Endothelial Cells (HDMEC; Lonza, Walkersville, MD, USA) were cultured in Endothelial Cell Growth Medium-2 (EGM2-MV; Lonza). To induce vasculogenic differentiation, SHED or DPSC were exposed to EGM2-MV supplemented with 50 ng/mL rhVEGF165 (R&D Systems, Minneapolis, MN, USA). In selected experiments, cells were exposed to 0 or 25 ug/mL anti-VEGF antibody, i.e. bevacizumab (Genentech, San Francisco, CA, USA).
7
Stem Cell Culture and Stimulation
8
Cytokine Modulation of NK Cell Chemokine Receptors
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NK cells were freshly isolated from healthy donor PBMCs (n = 3 donors) using Rosette Sep (Stem Cell Technologies, Vancouver, BC, Canada). Fresh NK cells were resuspended in NK media without IL-2 at a concentration of 5 × 105 cells/mL in a 24-well cell culture plate (Corning). Next, 20 ng/mL rh (recombinant human) G-CSF (Amgen, Thousand Oaks, CA, USA), 200 U/mL IL-2, 50 ng/mL rhTNF-α (R&D Systems, Minneapolis, MN, USA), 200 ng/mL rhIFN-γ (R&D Systems), 20 ng/mL rhIL-4(R&D Systems), 20 ng/mL rhIL-15(R&D Systems), 20 ng/mL rhGM-CSF (R&D Systems), 20 ng/mL rhIL-10 (R&D Systems), 5 ng/mL rhTGF-β (R&D Systems), 50 ng/mL rhCCL5 (R&D Systems), 50 ng/mL rhCCL3 (R&D Systems), 50 ng/mL rhVEGF165 (R&D Systems), or 100 ng/mL rhSDF-1α (R&D Systems) were individually added to each well. NK cells in media with no cytokine were used as the control. All samples were incubated for 24 h in 37 °C 6.5% CO2. Cells from each sample were collected and stained for flow cytometry-based identification of CCR1, CCR5, CXCR4, and CXCR6 surface expression.
9
Endothelial Differentiation of DPSCs
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Confluent DPSCs at passage 3 were exposed to the endothelial induction medium for 7 days containing 2% FBS and different concentrations (0%, 5%, 10%, 20%, 50%, or 80%) of CGF, with or without 50 ng/mL of rhVEGF165 (R&D systems, Minneapolis, MN, USA; 293-VE-010/CF). Undifferentiated cells were maintained in DMEM containing 2% FBS as the negative control. The medium was refreshed every other day.
10
Vascular Differentiation of Dental Stem Cells
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