N1e 115 cells

Human embryonic kidney cell line HEK 293T cells (ATCC CRL-11268) and mouse neuroblastoma N1E-115 cells (ATCC CRL-2263) were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 50 U penicillin-streptomycin at 37°C with 5% CO₂ inside a humidified incubator. Transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Mouse neuroblastoma N1E-115 cells (ATCC, Bethesda, MD; passage numbers from 10 to 13) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM glutamine and 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries, Beit Haemek, Israel). Human neuroblastoma SH-SYS5 cells (ECACC, Public Health England, Porton Down, Salisbury, UK; passage numbers from 14 to 16) were maintained in Ham's F12: minimum essential media (MEM) Eagle (1:1), 2mM Glutamine, 1% non-essential amino acids, 15% fetal bovine serum (FBS) and 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries, Beit Haemek, Israel). The cells were incubated in 95% air/5% CO₂ in a humidified incubator at 37°C. N1E-115 cells were plated on 35mm dishes (81156, 60 μ-Dish, Ibidi, Martinsried, Germany) at a concentration of 25*10⁴ cells/dish and then were differentiated with reduced FBS (2%) and DMSO (1.25%) containing medium during five days before transfection and seven days before the experiment. On the day of the experiment, differentiated N1E-115 cells were treated for 1 hrs with zinc chloride (ZnCl₂; final concentration, 400 μM, Sigma, Rehovot, Israel) with or without NAP (10⁻¹²M). Cultured SH-SY5Y cells were plated in 10cm dishes at a concentration of 0.5*10⁶/dish and differentiated with retinoic acid at a concentration of 10 μM during seven days.

Cell viability was determined by MTT assay, as reported previously [41 (link)]. N1E-115 cells, the mouse neuroblastoma cell lines, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Briefly, N1E-115 cells cultured in 96-well plates were treated with DMSO or various concentrations of TS-157 (0.1, 0.3, 1, 3, 10, 25, 50, 100 μM) for 48 h in DMEM. Then cells were incubated with MTT (5 mg/mL) for 4 h at 37 °C in humidified atmosphere of 5% CO₂ and 95% air at 37 °C. The medium with MTT was then removed, DMSO was added to each well, and absorbance was measured at 490 nm in a TECAN (Tecan, Shanghai, China) plate reader.

C57BL/6 (wild-type, WT) mice were purchased from Charles River, Sulzbach, Germany. Unc93b1^-/- mice were bred and maintained under specific-pathogen-free conditions at the animal facility of the Helmholtz Centre for Infection Research Braunschweig, Germany, and are described in (22 (link)). Animals were maintained and handled in accordance with the guidelines of the committee for animal care, the German Animal Protection Law, and approved by the Regional Office for Health and Social Services in Berlin (Landesamt für Gesundheit und Soziales – LAGeSo, Berlin, Germany).
N1E-115 cells, SH-SY5Y cells, and HMC3 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS, vol/vol) and 1% penicillin/streptomycin. The oligodendroglial precursor cell line Oli-neu was generously provided by Dr. J. Trotter [Institute of Molecular Biology, Johannes Gutenberg-University, Mainz, Germany (23 (link))] and was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (all obtained from Invitrogen, Darmstadt, Germany). Cells were grown at 37°C in humidified air with 5% CO2.

N1E-115 cells, the mouse neuroblastoma cell lines, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Undifferentiated N1E115 cells were cultured in DMEM complete medium with 10% fetal bovine serum, 100 U mL⁻¹ of penicillin, and 100 μg mL⁻¹ of streptomycin and maintained at 37 °C in a humidified incubator supplemented with 5% CO₂. N1E-115 cells were seeded at a density of 1 × 10⁴ cells per mL on poly-l-lysine-coated 96-well plates and grown with DMSO or different tested compounds (10 μM) in DMSO solution for four days. Four days after incubation, morphometric analysis was performed on digitized images of live cells taken under phase contrast illumination with an inverted microscope linked to a camera. Images of five fields per well were taken with an average of 100 cells per field. Cells that had at least one neurite with a length that was twice as long as the body diameter were expressed as a percentage of the total cells in the field. The counting was performed in a blinded manner. All experiments were performed at least five times.