Penicillin streptomycin

The cell line Oli-neu was cultured in modified Sato media [28 (link)]. Cell culture dishes were coated with poly-L-lysine (Sigma). HEK293T cells were cultured in DMEM (Sigma) with 10% Horse serum (Biochrom) and 1% Sodium-pyruvate (Sigma). Transfection of HEK293T cells with the NG2del constructs was effected by a standard protocol using the GenePulserXcell (Bio-Rad). Transcription was increased by including 4 mM sodium butyrate for the protease assay.
Cerebella of postnatal day 8–9 homozygous NG2-EYFP(NG2-KO) mice [29 (link)] or C57BL/6N mice (as control) were dissociated in 1% trypsin, 0.05% DNase in HBSS using a fire-polished Pasteur pipette to obtain a single-cell suspension, followed by seeding on poly-L-lysine-coated dishes. The cells were cultured in B27 medium containing DMEM, pyruvate, triiodo-L-thyronine, L-thyroxine (Sigma), B27 supplement (Gibco), 10ng/ml PDGF, 5ng/ml FGF (PrepoTech) and 1% HS. The medium was changed on the following day and renewed every 3–4 d. After 10-14d (after morphological assessment) cultures were stressed with H₂O₂ in B27 medium without growth factors.
Glioblastoma cells (R10) were cultured on ECM (Sigma) gel-coated dishes. ECM Gel was diluted 1/10 with Neurobasal media. Growth medium was Neurobasal medium (Invitrogen) with N2 supplement, B27 supplement, L-glutamine, EGF, FGF and 1% [v/v] penicillin/streptomycin (Serva).

The mouse motor-neuron-like hybrid cell line (NSC34), kindly provided by Dr. N.L. Cashman (University of British Columbia, Vancouver, CAN) [99 (link)], was maintained in DMEM medium supplemented with 5% fetal bovine serum (FBS)(F7524, Sigma-Aldrich), 1 mM L-glutamine (ECB3004D, Euroclone, Pero, Italy), and penicillin-streptomycin (penicillin, 31749.04, SERVA Electrophoresis GmbH, Heidelberg, Germany; streptomycin, 35500.01, SERVA Electrophoresis GmbH), at 37 °C in 5% CO₂. For experiments involving testosterone treatment, a dextran-treated charcoal-stripped FBS was used to selectively remove hormones [100 (link),101 (link)]. For the expression of the exogenous mutant proteins, NSC34 cells were transiently transfected with 0.7 μg of plasmid DNA using transferrin (T8150, Sigma-Aldrich) in conjunction with Lipofectamine reagent (18324012, Thermo Scientific Life Sciences Research, Waltham, MA, USA) to improve transfection efficiency.