Dmem f12 ham medium

Cell lines were obtained from ECACC, apart from BCH‐N‐DW, which is a novel neuroblastoma cell line derived from a bone marrow biopsy (Table S1; C. McConville, unpublished data), SHEP Tet‐21/N which was a kind gift from Prof. M. Schwab, and SK‐N‐AS MYCN‐ER which was a kind gift from Prof. A Sala. Cell lines except SHEP Tet‐21/N and SK‐N‐AS MYCN‐ER were cultured in DMEM/F12‐HAM medium (Sigma, Gillingham, Dorset, UK) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L‐glutamine, and 1% non‐essential amino acids (Sigma) at 37°C in a 5% CO₂ incubator. SH‐EP TET‐21/N cells 35 were cultured in RPMI1640 medium (Invitrogen, Paisley, UK) containing 10% tetracycline‐free FBS (Bioclear, Calne, Wiltshire, UK), with other additives as above and for MYCN silencing 1 μg/ml tetracycline (Sigma) was added. SK‐N‐AS MYCN‐ER cells 36 were cultured in DMEM/F12 containing 10% charcoal‐stripped FBS (Appleton Woods, Birmingham, UK) with other additives as above and for MYCN induction 400 nM 4‐hydroxytamoxifen (Sigma) was added. Neural crest cells were cultured as described previously 37.
Neuroblastoma tumor samples (Table S2) were obtained from Bristol and Birmingham Children's hospitals with appropriate local ethical approval and used as specified in the UK Human Tissue Act.

Primary MPM cultures were established from surgical specimens and maintained with serum and without serum as previously described [25 (link),49 (link)]. MPM cells-SPC111, SPC212, ZL34 and ZL55, were established in our laboratory [50 (link)] and were maintained in DMEM:F12 (Ham) medium (Sigma-Aldrich, St. Louis, MO, USA) with 15% foetal calf serum (FCS, Invitrogen/GIBCO) and 1% penicillin/streptomycin (Sigma-Aldrich); Mero- 14, Mero-41, Mero-48, Mero-82, Mero-83, Mero-84 and Mero-95 were maintained in DMEM:F10 (Ham) medium (Sigma-Aldrich) with 15% FCS and 1% penicillin/streptomycin; ONE-58, ACC-Meso-1 and ACC-Meso-4 were maintained in RPMI-1640 (Sigma-Aldrich) with 15% FCS and 1% penicillin/streptomycin; MSTO-211H, H2052, H2452, H226, NO36 and H596 cells were maintained in RPMI-1640 with 10% FCS and 1% penicillin/streptomycin. ZL55SPT and SDM103T2 primary cells were grown in serum-free medium [25 (link)]. Normal mesothelial cells NP3, SDM77, SDM85, SDM104, SDM58 and SDM71 were cultured as previously described [51 (link)]. NP3 cells were also grown in serum-free medium. To induce growth arrest, cells were treated either with Hedgehog signaling inhibitor (HhAntag) [25 (link)] or with dual PI3K/mTOR inhibitor (NVP-BEZ235, Novartis, Switzerland) [26 (link)], as previously described.

MDA-MB-231 (ECACC 92020424) cells were expanded by culture in complete culture medium (CCM) prepared from Dulbecco’s Modified Eagle’s Medium (#D8437, DMEM/F12/Ham medium, Sigma-Aldrich, North Ryde, NSW, Australia) supplemented with 10% fetal bovine serum (#12003C, FBS; Sigma-Aldrich, North Ryde, NSW, Australia) and 1% penicillin–streptomycin (PS; 10,000 U/mL; #15140122, Gibco) under standard conditions (37 °C, humidified, 5% CO₂ gas atmosphere). The culture medium was changed every two days, and the cellular growth was controlled using a phase-contrast microscope and cell counting. According to the cell counting data, the average population doubling time of MDA-MB-231 cells (used in the experiments in passages 4 to 6) was approximately 34 h, with an average viability of ~98%. The same culture medium was used for all in vitro experiments, unless otherwise specified.

HGSOC mouse orthotopic cell lines; 30200 and 60577 were derived from serous ovarian cancer genetically engineered mouse models (GEMM; ref. 15) and engineered to express Trp53^−/−, Brca1^−/−, and Rb inactivation. Following intraperitoneal cell injection, they form extensive disease in the omentum and also metastasize to the spleenoportal fat, lesser omentum, and mesentery. The two models vary in their average survival time with 20 weeks (30200) and 6 weeks (60577). HGS2 cell line was derived from a Pax-8-cre inducible GEMM driving inactivation of Trp53, Brca2, and Pten in the fallopian tube epithelium. This model is syngeneic with C57 BL/6J background and upon intraperitoneal cell injection form extensive disease in the omentum and peritoneum similar to the other models by 12 to 14 weeks.
These orthotopic transplantable lines were grown in DMEM/F12 Ham medium (Sigma-Aldrich) constituted with 2% FBS (Gibco-Invitrogen), 100 units/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate (Invitrogen). The medium was also supplemented with 5 mL of 100× Insulin/Transferrin/Selenium (Invitrogen), 5 mL of 50 µg/mL of Hydrocortisone (Sigma-Aldrich), 5 mL of 100x anti-anti (Gibco), and 500 µl of 10 µg/mL murine EGF. Cells were trypsinized with 0.25% trypsin-EDTA (Sigma-Aldrich) and split 1:4.

BCAA were prepared as a mixture of leucine, isoleucine and valine at 0.2‐12 mmol/L each. BCAA, rapamycin, wortmannin, diphenyliodonium chloride (DPI), L‐NAME, acetylcholine, phenylephrine and DEA‐NO were obtained from Sigma‐Aldrich (Sigma Chemical Co., St. Louis, MO, USA). DMEM‐F12 Ham medium and foetal bovine serum (FBS) were also obtained from Sigma‐Aldrich. 5‐Aminoimidazole‐4‐carboxamide‐1‐d‐ribofuranoside (AICAR) was purchased from Toronto Research Chemicals (North York, Canada), while BAY‐11‐7082 and ML171 were obtained from Calbiochem (La Jolla, CA). Mito‐TEMPO was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Celecoxib was a kind gift from Pfizer Inc. (Groton, CT, USA) and gp91dstat was obtained from Anaspec (Fremont, CA). wortmannin and BAY‐11‐7082 were dissolved in DMSO. ML171 was dissolved in 75% ethanol. Further dilutions were in distilled water.

CD24⁻ and CD24⁺ single cell suspensions were prepared and plated in nonadherent conditions at 600 cells/cm² in DMEM F12 HAM medium (Sigma, Rehovot, Israel) containing 20 ng/ml bFGF (Sigma), 20 ng/ml EGF (Sigma), 4 μg/ml of Heparin (Sigma) and B-27 supplement (1:50 dilution, GIBCO, Burlington, ON, USA), and cultured at 37 °C with 5 % CO2. Tumorsphere-forming efficiency (percentage) was calculated after 5 days as follows: (number of tumorspheres (>50 mm in diameter) per well/number of cells seeded per well)*100. To assess self-renewal, primary tumorspheres were centrifuged at 115 × g for 5 minutes, the pellet was resuspended in 300 μl of 0.5 % trypsin/0.2 % EDTA for 3 minutes at 37 °C. Tumorspheres were disaggregated into single cell suspension with the use of a 25-G needle and syringe, (trypsin was neutralized with medium containing serum). Cells were centrifuged at 580 × g for 5 minutes, the pellet was resuspended in ice-cold PBS, and single cell suspension was assured under a microscope. Single cells were plated at the same seeding density that was used in the primary generation. Tumorspheres (>50 mm in diameter) were measured after 5 days in culture. Self-renewal was calculated by dividing the number of secondary tumorspheres formed by the number of primary tumorspheres formed.

Isolation of the DRG neuron was performed as previously described18 (link) with a few modifications. The DRG from the thoracic and lumbar spinal cord of 1–3 day-old rats were cut in small pieces and incubated for 1h at 37 °C in a solution containing 124 mM NaCl, 5 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM MgSO₄, 2.4 mM CaCl₂, 24 mM NaHCO₃, 10 mM Glucose, 1.6 mg/ml collagenase type II (Sigma), and 1.6 mg/ml trypsin (Difco Laboratories). They were gently triturated with a fire-polished glass pipette and the resulting solution was centrifuged at 800 rpm for 2 min. The obtained pellet was resuspended in DMEM-F12 HAM medium (Sigma) containing 10% FBS, penicillin and streptomycin. Cells were plated onto dishes coated with poly-L-lysine (Sigma). The cells were cultured in the DMEM-F12 HAM medium containing 10% FBS at 37 °C under 5% CO₂. All animal protocols were approved by the Animal Experiment Committee in the Graduate School of Bioagricultural Sciences of Nagoya University.