Dmem f 12 nutrient mix

SF188 and KNS42 paediatric GBM cell lines were cultured in Dulbecco Modified Eagle’s Medium (DMEM)/F-12 nutrient mix (Gibco) supplemented with 10% Foetal Bovine Serum (FBS) and 2% L-glutamine. Adult GBM cell line, GIN28 was cultured in DMEM (Gibco; Milton Park, Oxford, Innovation Centre 99 Park Drive Milton Park, Oxford OX14 4RY, UK) containing 15% FBS, 1% L-glutamine and antibiotics (1% penicillin and streptomycin). HeLa cell line were used as positive control and grown in DMEM (Gibco) with 10% FBS and 2% L-glutamine. Cells were passaged at approximately 80% confluency by dissociating with trypsin-EDTA and seeded onto treated tissue culture plastic (polystyrene) and chamber slides. GBM cell lines were passaged twice a week to maintain exponential growth at varying seeding ratios of 1:3–1:5 and at cellular densities of 15,000–30,000 cells per well. HeLa cells were passaged daily due to their rapid doubling time and plated at 5000 cells per well. Optimal culture conditions of 37 °C, 21% Oxygen and 5% Carbon IV Oxide was maintained for all experiments.

TUBB3–2A-mCherry iPSCs were co-transduced with a lentivirus encoding M2rtTA and the indicated tetO-cDNA. Cells were transduced in mTesR with 10 μM Rock Inhibitor. The following day, the medium was changed to neurogenic medium (DMEM/F-12 Nutrient Mix (GIBCO, 11320), 1x B-27 serum-free supplement (GIBCO, 17504), 1x N-2 supplement (GIBCO, 17502), and 25 μg/mL gentamicin (Sigma, G1397)) supplemented with 0.1 μg/mL doxycycline. Cells were sorted after 2 or 3 days of transgene expression using a SH800 FACS Cell Sorter in semi-purity mode. Sorted cells were replated onto matrigel-coated 24-well plates and cultured in neurogenic medium supplemented with 10 ng/mL each of BDNF, GDNF and NT-3 (PeproTech) until harvest after 6 or 7 days.
Total RNA was extracted using RNeasy Mini Kit (QIAGEN) and 100 ng of RNA was used to develop RNA-seq libraries. RNA-sequencing libraries were prepared using the Truseq Stranded mRNA kit (Illumina) according to the manufacturer’s protocol. The libraries were sequenced on a NextSeq 500 on High Output Mode with 75 bp paired-end reads.

Tumorspheres were established from NSCLC tissue surgically removed from lung cancer patients at Odense University Hospital. The Regional Ethics Committee of the region of Southern Denmark approved the protocol (S-20140170). As described in [33 (link)], the primary cells were prepared and grown in non-adherent flasks in serum-free medium at 37 °C in a humidified atmosphere with 5% CO₂. The serum-free medium consisted of DMEM/F12 nutrient mix, Glutamax^TM supplemented with 1% penicillin/streptomycin, 1% B27, 20 ng/mL epidermal growth factor (all from Thermo Fisher Scientific, Roskilde, Denmark, Roskilde, Denmark), and 20 ng/mL basic fibroblast growth factor (Peprotech, Stockholm, Sweden)). A graphic overview of the experiments are presented in Figure 9.