Platelet derived growth factor

GABA, Bicuculline methochloride, Tetrodotoxin citrate, NBQX and D-APV were purchased from Abcam Biochemicals (Cambridge, UK); Furosemide (100 mM in dimethyl sulfoxide (DMSO)), Bumetanide (50 mM in EtOH) and PP2 (10 mM in DMSO) were purchased from Tocris Biosciences (Bristol, UK); FluoZin-3AM (Invitrogen, 5 mM DMSO); K252a (Calbiochem, Merck S.p.a., Milano, Italy); DIOA (Santa Cruz Biotechnology, Inc., Heidelberg, Germany; 200 mM in EtOH); BDNF (PeproTech EC Ltd., London, UK); TPEN (10 mM in EtOH), Gramicidin (50 mg/ml in DMSO), Sulfasalazine (500 mM in DMSO), platelet-derived growth factor and all chemicals used were purchased from Sigma-Aldrich, Milan, Italy. Where not indicated, all drugs were dissolved in water. Antibodies were purchased and used as follows: rabbit anti-Phospho TrkB (phospho Y515, 1 : 500, Abcam, Cambridge, UK), rabbit anti-Phospho Src (phospho Y416, 1 : 2000, Abcam), mouse anti- Neuronal Class III β-tubulin (TuJ1, 1 : 2000, n.cat MMS-435P, Covance, San Diego, CA, USA), rabbit anti-actin (1 : 5000, Sigma-Aldrich); rabbit anti-KCC2 (1 : 1000; Millipore, Temecula, CA, USA).

The primary antibodies MAPK (Erk) (#9102, 1:1000), Akt (1:1000, #9272), Phospho-EGF Receptor (Tyr1068) (1:1000, #2234), p(Thr308)-Akt (1:1000, #9275), Phospho-Akt (Ser473) (1:1000, #9271), Phospho-MAPK (pERK) (1:1000, #9101), β-Actin (1:10000, #4967) were purchased from Cell Signaling (Leiden, The Netherlands) and anti-EGFR (1:1000, sc-03-G) was purchased from Santa Cruz Biotechnology(Texas, USA). Cetuximab (ERBITUX) was ordered from Merck (Dietikon, Switzerland). Gefitinib (Iressa) was bought from Sigma (Zwijndrecht, The Netherlands); sunitinib was purchased from LC Laboratories (Woburn, USA). Entinostat and SAHA were purchased from Selleckchem (Munich, Germany). Staurosporine and cisplatin were purchased from Sigma-Aldrich (Zwijndrecht, Nederland). Doxorubicin was purchased from Teva Pharmaceuticals. All drugs were aliquoted in DMSO and stored at -20°C. The human epidermal growth factor (hEGF) and platelet-derived growth factor (PDGF) were purchased from Sigma-Aldrich (Zwijndrecht, Nederland).

Platelet derived growth factor (PDGF) was purchased from Sigma (St. Louis, MO), and OPN (sc-21742) and β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology Inc. (Beverly, MA). Horseradish peroxidase (HRP)-conjugated IgG antibody (Santa Cruz Biotechnology Inc.) was used as the secondary antibody. PCR primers were from Bioneer (Seoul). AP-1 (10024-2-AP) antibody was purchased from Proteintech (Proteintech Group, Chicago, USA), and C/EBPβ (ab15049) antibody from Abcam (Cambrige, MA). Restriction enzymes were supplied by Promega (Madison, WI).
AP-1 and C/EBPβ siRNA oligonucleotides were synthesized by Bioneer (Daejeon, Korea). siRNA molecules were transfected into cells using Lipofectamine 2000 siRNA transfection reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. siRNA sequences against AP-1 and C/EBPβ were as follows: AP-1, ACUGUAGAUUGCUUCUGUA (sence) and UACAGAAGCAAUCUACAGU (antisense); C/EBPβ, GACAAGCUGAGCGACGAGU (sence) and ACUCGUCGCUCAGCUUGUC (antisense).

MPCs were seeded at 1 × 10³ cells/cm² on tissue culture plastic or NFs and differentiated neurotrophically using a modified 10-day protocol [27 (link), 29 (link), 42 (link)]. Twenty-four hours after seeding, MPCs were incubated with GM supplemented with 10 mM β-mercaptoethanol (Sigma) for 24 h, followed by 48 h in GM supplemented with 10 mM β-mercaptoethanol + 35 ng/ml retinoic acid (Sigma). For the 6 following days, MPCs were incubated with neurotrophic medium (DMEM/Ham’s F12 + PSF (Invitrogen) supplemented with 2% FBS, 2% B-27 (Invitrogen), 6 mg/ml retinoic acid, 1 ng/ml FGF-2 (Sigma), 10 ng/ml platelet-derived growth factor (PDGF; Sigma), 150 ng/ml heregulin (an isoform of neuregulin-1; Sigma), and 10 μM forskolin (Sigma)).
Following neurotrophic differentiation, MPCs (designated nMPCs) were washed with PBS and either lysed with TRiZol, or incubated with basal medium (see above) for 48 h to produce conditioned medium (nMPC-CM). For DRG coculture experiments, tissue culture plastic-cultured nMPCs were trypsinized and transferred to DRG-containing fibers at a concentration of 1 × 10³ cells/cm².