Recombinant human pdgf aa

Cell culture reagents were purchased from Sigma (Dorset, UK)—Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium Eagle (MEM), l-glutamine, poly-l-lysine (PLL), papain, NAA—or Life Technologies (Paisley, UK)—fetal bovine serum (FBS), penicillin–streptomycin (pen–strep), trypsin–EDTA, phosphate buffered saline (PBS). ¹³C-labelled compounds were obtained from Cambridge Isotope Laboratories, MA, USA. The mass spectrometry derivatization reagents MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide), MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) and the t-BDMS-Cl (tert-butyldimethylchlorosilane) were purchased from Regis Technologies, Inc. (Morton Grove, IL, USA). Recombinant human PDGF-AA and Recombinant human FGF-basic were purchased from PeproTech (Rocky Hill, NJ). All other chemicals were of the purest grade available from Sigma (Dorset, UK).

A high-grade glioma cell line was developed using a PDGF-internal ribosomal entry site (IRES) retrovirus implanted into the cerebral white matter of p53^−/− PTEN^−/− mice^{26 (link)}. Tumor cells were then isolated and propagated in adherent conditions on polylysine-coated flasks maintained in culture using a basal media DMEM (Thermo Scientific, Waltham, MA, USA) with 0.5% FBS (Thermo Scientific, Waltham, MA, USA), antibiotic–antimycotic (Thermo Scientific, Waltham, MA, USA), N2 supplement (Thermo Scientific, Waltham, MA, USA), and supplemented with recombinant human PDGF-AA (Peprotech, Rocky Hill, NJ, USA) and FGFb (Peprotech, Rocky Hill, NJ, USA) both to a concentration of 10 ng/ml.

The cell population derived from normal human astrocytes (NHA) and P3#GBM cells were kind gift from the Department of Biomedicine at the University of Bergen (Bergen, Norway). U87MG, U251 and A172 were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). P3#GBM cells were cultured in serum-free Neurobasal medium (Gibco, USA) supplemented with 2% B27 Neuro Mix (Thermo Fisher Scientific, USA), 20 ng/mL epidermal growth factor (EGF; Thermo Fisher Scientific, USA), and 10 ng/mL basic fibroblast growth factor (bFGF; PeproTech, USA). GBM and NHA cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies-Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies-Thermo Fisher Scientific, USA) and maintained at 37 °C in a humidified chamber containing 5% CO₂. Recombinant human PDGF-AA (Peprotech, USA) was dissolved in phosphate buffered saline (PBS), and AG-1296, an inhibitor of PDGFRα (Selleck, China), was dissolved in DMSO before addition to media. The small molecule MK-2206 (Apexbio, USA) was dissolved in DMSO and used as an inhibitor of AKT phosphorylation.