Nha cells

The glioma cell lines U87ΔEGFR and GL261 were seeded on tissue culture dishes (BD Falcon, Franklin Lakes, NJ, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U penicillin, and 0.1 mg/ml of streptomycin. GL261 cells were provided by Dr. A. Natsume, Nagoya University (Nagoya, Japan). NHA cells were purchased from Takara Bio Inc. (Shiga, Japan).

NHA cells (TaKaRa, Japan) were cultured in astrocyte growth medium (Lonza, Walkersville, MD, USA). To create an experimental human glioma model, NHA cells were introduced with genes for hTERT (T), SV40ER (S), H-RasV12 (R), and myrAKT (A), as previously reported (20). The cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Cansera, Ontario, Canada). After washing the culture dish with cold phosphate buffered saline (PBS), the cells were scraped into cold PBS containing 10 mM EDTA and collected by centrifugation at 1,000 × g for 3 min. The pellet was washed four times with phosphate-buffered saline (PBS) to remove fetal bovine serum (FBS), thereby minimizing contamination from FBS-derived glycoproteins. The cells were stored at −30°C as pellets (1 × 10⁶ cells/pellet). The total cellular protein concentration was determined by using the bicinchoninic acid (BCA) method (Pierce Biotechnology, Rockford, IL, USA).