Mem vitamin solution

For amino acid deprivation experiment, basal medium was prepared from Earle's balanced salt (EBS: Sigma E2888) supplemented with MEM vitamin solution (Sigma M6895), 0.11 g/L sodium pyruvate (Sigma P4562) and 0.0001 g/L Fe(NO₃)₃9H₂O (Sigma 216828). To this basal medium, we added each amino acid in the same concentration as amino acids contained in DMEM (Table 1). After we determined the maximal expression of GADD34 was achieved with Tyr/Cys-deprivation, we used Tyr/Cys-deprivation medium in DMEM, which was made by Cell Science and Technology Institute, Inc.

We used primary BTICs that were isolated from freshly resected human gliomas, as described (10). Specimen sampling and BTIC culture were approved by the Ethics Committee of the University of Regensburg (No° 09/101) and all patients gave written informed consent. BTICs were kept in DMEM low glucose medium (DMEM with 1 g/L of glucose; Sigma-Aldrich, St. Louis, MO, USA, #D6046) containing Epidermal Growth Factor (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-097-751) and Fibroblast Growth Factor (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-093-842) supplemented with 50 U (v/v) Penicillin, 0.05% (v/v) Streptomycin (#P4333), 2 mM (v/v) L-Glutamine (#G7513), 1% (v/v) MEM Vitamin Solution (#M6895), and 1% (v/v) non-essential amino acids (#M7145) (all Sigma-Aldrich, St. Louis, MO, USA). For differentiation, growth factors were withdrawn, and cells were exposed to 10% fetal calf serum (FCS) for at least two weeks. Cells were incubated at 37 °C, 5% CO₂, 95% humidity in a standard tissue culture incubator.

Madin-Darby Canine Kidney (MDCK) cells were purchased from the American Type Culture Collection (ATCC) and maintained in Modified Eagle’s Medium (MEM; Corning, USA) containing 5% heat-inactivated fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco), and 1% MEM vitamin solution (Sigma, USA). Splenocytes from immunized mice were cultured in Roswell Park MEMorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.

For the experimental diets, a vitamin mixture, cellulose powder, and corn starch were purchased from Oriental Yeast, and soybean oil was purchased from Nacalai Tesque. For cell culture medium, Earle's buffered salt solution (EBSS) and MEM vitamin solution were purchased from Sigma Aldrich. Dulbecco's modified Eagle's medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Nissui Pharmaceutical Co. Fetal bovine serum (FBS) was purchased from Sigma–Aldrich. Penicillin and streptomycin were purchased from Banyu Pharmaceutical Co.

C2C12 (CRL-1772, ATCC) and Rhabdomyosarcoma cells (RD, CRL-136, ATCC) were cultured in DMEM (Gibco). C2C12 was supplemented with 10% FBS, 2mM L-glutamine (Sigma-Aldrich), 1% penicillin/streptomycin while RD was supplemented with 2% MEM vitamin solution and 2% MEM non-essential amino acids (Sigma-Aldrich). Cells were maintained in a 5% CO₂ atmosphere at 37°C. The culture medium was changed every 3 days.
To induce myoblast differentiation, C2C12 cells were changed to differentiation medium containing DMEM 2% HS [16 (link)] when they reached 80% confluence. In the case of RD, cells were changed to differentiation medium plus 2 μM insulin (Sigma-Aldrich) [17 (link)] and, after 24 h 5 μM of retinoic acid (Sigma-Aldrich) was added. The cells were harvested at different time points until 6 days of differentiation.
For the induction of glycolytic metabolism in C2C12, the cells were cultured in DMEM with glucose at high concentration (30 mM, HG) or treated with 30 μmol/L berberine (Sigma-Aldrich) as previously described [18 (link)–19 (link)]. Additionally, the induction of glycolytic metabolism was performed by decreasing oxygen levels up to 2% during 24 and 48 h.
When necessary, C2C12 cells were induced to differentiate and treated with 2 ng/ml leptomycin B (LMB, Sigma-Aldrich) for 24 h before fixed or harvested [20 (link)].