2.2. Cell cultures and medium composition

Cells were obtained from the following sources: HeLa and HepG2 cells were purchased from JCRB (Osaka, Japan); BxPC-3, HT1080, and G361 cells were from ECACC (Salisbury, Scotland); A375-P, A375-MA2, and MDA-MB-231 cells were from ATCC (Manassas, VA, USA). HaCaT cells were provided by Dr. Fusenig (German Cancer Research Center, Heidelberg). MCF-7 cells were a kind gift from Dr. Kametani (Tokai University School of Medicine, Kanagawa, Japan). All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare, South Logan, UT, USA), 50 U/mL penicillin, 50 mg/mL streptomycin, and non-essential amino acids (Life Technologies, Carlsbad, CA) at 37 °C and 5% CO₂. For detachment experiments, tissue culture dishes or plates were coated with 3 mg/mL poly-HEMA in 95% ethanol as mentioned previously [18] with some modifications, and were then incubated at 37 °C for more than a day until dry. Media deficient for glucose, glutamine, or both were made based on powdered glucose- and glutamine-free DMEM (Sigma-Aldrich) that contained neither phenol red nor pyruvate. When these nutrients had to be present, glucose or glutamine were added into the nutrient-deficient medium to a final concentration of 5.5 mM and 2 mM, respectively. All nutrient-deficient DMEM was prepared by dissolving inorganic salts (CaCl₂ 265 mg/L, MgSO₄ 97.67 g/L, KCl 400 mg/L, NaHCO₃ 3.7 g/L, NaH₂PO₄ 109 mg/L, NaCl 6.8 g/L, Fe(NO₃) ₃·9H₂O 0.1 mg/L) in ultrapure water, with the final pH adjusted to pH 7.4. To examine the nutrient requirements, nutrient-deficient culture media with or without a specific nutrient were changed during detachment culture conditions. To test for auxotrophy under the adherent state, the intended medium was exchanged after the cells were sufficiently attached. Media for all experiments were supplemented with 10% dialyzed FBS.