Neurotrophin 3

Tissue from neuroblastoma PDXs was dissociated and digested for 45 min at 37 °C with Liberase (0.15 mg/mL, Roche), passed through a 70 µm cell strainer (BD Biosciences) and cultured in stem cell medium (SC medium) at 37 °C in 5% CO₂^{10 (link)}. Spheres were dissociated using Accutase (Sigma-Aldrich, St. Louis, MO). PDX #2 had been established from cerebral metastasis from a stage 4 tumor where the patient had undergone prior treatment while PDX #3 had been established from a primary stage 3 tumor in the adrenal gland. For serum culture, SC medium excluding growth factors and B-27 supplement was supplemented with fetal bovine serum (FBS) or 10% charcoal-stripped FBS (Thermo Scientific). For monolayer culture on human recombinant laminin (Biolamina), plates were coated according to manufacturers instructions. PDX cells were routinely grown to confluence and dissociated using Accutase. The neuroblastoma cell line SK-N-BE(2)c (ATCC) was cultured as previously described^{38 (link)}. All cells were routinely screened for mycoplasma and authentication was performed by SNP profiling of SK-N-BE(2)c cells, PDX cells and corresponding PDX (Multiplexion, Germany).
Treatment with MYC-MAX inhibitor 10058-F4 (Sigma) was performed for 72 h at 60 μM or 75 μM. Treatment with neurotrophins was done for 4 days with nerve growth factor (NGF, 50 ng/ml, PeproTech), neurotrophin-3 (NT-3, 50 ng/ml, PeproTech), or a combination. Prior to neurotrophin treatment, cells were cultured in SC medium, 2% or 10% serum for 7 days. For cytostatic treatments, cells were treated with cisplatin, doxorubicin or etoposide in BRANDplates® 96-well plates (VWR) directly after seeding or 48 h post-seeding. CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to determine cell viability 72 h post-treatment. Cell culture images were captured with a Carl Zeiss AxioCam IC microscope and the ZEN software.

Differentiation of NGN2-ESCs was performed according to Wang et al. (31 ), with some modifications. Briefly, to produce mature and terminally differentiated human neurons from NGN2-expressing hESCs, the cells were cultured in doxycycline to initiate differentiation and proliferation of neuronal precursors. This was followed by culture in terminal differentiation medium to generate mature and nondividing BioID2-expressing neurons for identification of interacting proteins by proximity biotinylation. The initial 5 days in culture expanded the numbers of immature neurons available for terminal differentiation and immediate use or for cryopreservation. Terminal differentiation in culture for 2 weeks or more produced mature nondividing neurons.
ESCs were dissociated with Accumax (Sigma) and plated (2 × 10⁷ cells/10 cm dish) on Matrigel in predifferentiation medium consisting of Dulbecco’s modified Eagle's medium/F12, doxycycline (1 μg/ml), nonessential amino acids (1×), laminin (0.2 μg/ml), brain-derived neurotrophic factor (10 ng/ml), neurotrophin-3 (10 ng/ml), and Y-27632 (10 μg/ml). The media were changed daily, and Y-27632 was removed after 48 h. On the fifth day of differentiation, the immature neurons were gently dissociated in Accumax and plated on Matrigel-coated 10 cm dishes (2 × 10⁷ neurons/dish) in 20 ml differentiation medium containing 1:1 Dulbecco’s modified Eagle's medium/F12:Neurobasal-A, nonessential amino acids, Glutamax (1×), doxycycline (1 μg/ml), laminin (1 μg/ml), B-27 supplement (0.5×), N-2 supplement (0.5×), brain-derived neurotrophic factor (10 ng/ml), and neurotrophin-3 (10 ng/ml). One-quarter of the medium was changed every week.

The hippocampus was excised. Total RNA was extracted from the hippocampus using a RNeasy Lipid Tissue Kit (QIAGEN Sciences Inc.) and transcribed into cDNA with random primers using Takara PrimeScript RT Master Mix (Takara, Osaka, Japan). For quantitative PCR, we amplified the cDNA using a LightCycler 96 System (Roche Diagnostics Co., Mannheim, Germany) with THUNDERBIRD qPCR Mix (Toyobo Co., Osaka, Japan). Primer sets specific for mouse brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 2 (IGF2), and vascular endothelial growth factor (VEGF) were prepared in accordance with the manufacturer's instructions. The primer sequences are shown in Supplemental Table S1. The reactions were cycled 45 times with denaturation at 95 °C for 10 s, and with annealing and elongation at 65 °C for 60 s each. The relative expression level of each mRNA was normalized using the mRNA level of β-actin.