U87 atcc htb 14

293T cells, and the GBM cells, U87 (ATCC #HTB-14) and A172 (ATCC #CRL-1620), were obtained from the American Type Culture Collection (ATCC). The U87 cell line used in the present study is considered is probably a GBM cell line; however, its true origin is unknown. All cell lines were authenticated by STR profiling. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone; Cytiva). The culture medium was supplemented with 10% fetal bovine serum (HyClone; Cytiva), 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were cultured at 37°C with 5% CO₂ under a humidified condition. The A172R cell line was constructed by treating the A172 cells with stepwise increasing concentrations of TMZ (20 to 200 µM) for 4 months until a stable resistant phenotype was achieved (data not shown). These TMZ-resistant A172 cell populations were dispersed as single cells in 96-well plates and exposed to 200 µM TMZ to obtain TMZ-resistant subclones. Finally, 5 subclones were examined and they exhibited a similar morphology and growth rates. Thus, one subclone of these was designated as A172R cells and subjected to miRNA sequencing analysis. TMZ (#S1237) was purchased from Selleck Chemicals. Dimethyl sulfoxide (DMSO) was used to dissolve TMZ.

Human glioblastoma cell lines U87 (ATCC HTB-14, obtained from the American Type Culture Collection, Manassas, VA, USA), U251, LN18 and SF767 (obtained from C. Simon’s laboratory, UPENN, Philadelphia, PA, USA) were used and routinely maintained in Dulbecco’s Modified Eagle Medium (Lonza, Portsmouth, NH, USA) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO₂-humidified incubators as previously described. Cells were tested for mycoplasma contamination by PCR. When indicated, cells were treated with cobalt chloride (CoCl₂; Sigma, St Louis, MO, USA) at 100 μM (2 × 50 μM at 24 h and 48 h after transfection) or with MG132 (5 μM for 2 h; Sigma, St Louis, MO, USA) or irradiated at 5 Gy using the Gammacell 40 Exactor irradiator (Nordion, Ottawa, ON, Canada).