Cd140b

Diabetic media composed of EGM2 MV + 1% AGS supplemented with 30 mM D-glucose solution (Gibco, A2494001), 1 ng ml^-1 TNF-α (Novus Biologicals, 210-TA-005/CF) and 1 ng m⁻¹ IL-6 (Invitrogen, A42540) was applied from day 7 to day 14 or 28 to induce diabetic phenotypes. Diabetic media was newly prepared every 2 or 3 days and changed daily. EGM2 MV + 1% AGS supplemented with 30 mM D-Mannitol (Toronto Research Chemicals, TRC-M165000) in 1× PBS was used as an osmotic control, and EGM2 MV + 1% AGS only as an untreated control. Other treatments were prepared by supplementing EGM2 MV + 1% AGS with 10 μg ml⁻¹ CD140b (PDGFRB) Monoclonal Antibody (APB5, PDGFRβ inhibitor, eBioscience, 14-1402-82), 10 µM Imatinib Mesylate (STI571, PDGFRβ inhibitor, ApexBio, A1805), 10 µM TIE2 kinase inhibitor (ApexBio, A5979), 10 µM DAPT (γ-secretase inhibitor, Tocris, 2634), 30 µM 19,20-dihydroxydocosapentaenoic acid (DHDP) or 500 µg ml^-1 Advanced Glycation End product-BSA (AGE, Sigma-Aldrich, 121800-10MG). These treatments were applied from day 7 to 28 to alter pericyte-EC interactions, and fresh media were prepared every 2 days and changed daily.

Xenograft tumour tissue was cryo-sectioned (20 µm) and processed as previously described.^{6 (link)} Endothelial cells were stained with a monoclonal Armenian-hamster anti-mouse CD31 antibody (1:200, Abcam) or a fluorescein isothiocyanate (FITC)-labelled anti-CD31 antibody (1:200, clone 390, Biolegend), and apoptotic cells were detected with a rabbit anti-cleaved caspase-3 (Asp175) antibody (1:200, Cell Signaling). Pericytes were detected either by a rabbit anti-NG2 Chondroitin Sulphate Proteoglycan antibody (1:100, Millipore) or a rat anti-PDGF-R antibody (1:100, CD140b, eBioscience). Vascular endothelial (VE) cadherin was stained with a rat anti-CD144 (VE-cadherin) antibody (BD Pharmingen). The following secondary antibodies derived from goat were used to label the respective primary antibodies with fluorescent dyes: Alexa Fluor 647 anti-hamster, Alexa Fluor 594 anti-rat, Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 anti-hamster (1:500, Thermo Fisher Scientific). Vascular leakage was quantified by analysing intravenously injected FITC-dextran (molecular weight 2000 kDa, Sigma), pimonidazole adducts were detected with a FITC-labelled anti-pimonidazole antibody to visualise hypoxia; both procedures have been previously described in detail^{6 (link),19 (link)}; nuclei were stained with 4,6-diamidin-2-phenylindol (DAPI).