We performed gene expression
Selleckchem LY2874455 profiling of the cell populations treated with the same combinations of ATRA and LOX/COX inhibitors as in our previous experiments, and the results generate new knowledge about possible molecular mechanisms of the enhancement of ATRA-induced differentiation in neuroblastoma cells. Methods Cell lines and cell cultures SK-N-BE(2) (ECACC cat. no. 95011815) and SH-SY5Y (ECACC cat. no. 94030304) neuroblastoma cell lines were used for this study. Cell cultures find more were maintained in DMEM/Ham’s F12 medium mixture (1:1) supplemented with 20% fetal calf serum, 1% non-essential amino acids, 2 mM glutamine, and antibiotics: 100 IU/ml of penicillin and 100 μg/ml of streptomycin (all purchased from PAA Laboratories, Linz, Austria) under standard conditions selleck at 37°C in an atmosphere of 95% air: 5% CO2. The cells were subcultured 1-2 times weekly. Chemicals ATRA (Sigma Chemical Co., St. Louis, MO, USA) was prepared as a stock solution
at the concentration of 100 mM in dimethyl sulfoxide (DMSO; Sigma). CA (Sigma) and CX (LKT Laboratories, Inc., St. Paul, MN, USA) were dissolved in DMSO at the concentrations of 130 and 100 mM, respectively. Reagents were stored at -20°C under light-free conditions. Induction of cell differentiation Stock solutions were diluted in fresh cell culture medium to obtain final concentrations of 1 and 10 μM of ATRA, 13 and 52 μM of CA and 10 and 50 μM of CX. In all experiments, cells were seeded onto Petri dishes 24 h before the treatment,
and untreated cells were used as a control. The experimental design was the same as in our previous study [17]: cell populations were treated with ATRA alone or with ATRA and inhibitor (CA click here or CX) in respective concentrations. However, a combined treatment with 10 μM ATRA and 50 μM CX was not included in these experiments due to the predominant cytotoxic effect on cell populations. Cells were harvested after three days of cultivation in the presence of ATRA and inhibitors. Expression profiling Total RNA of treated cell populations was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma), and its concentration and integrity were determined spectrophotometrically. Conversion of experimental RNA to target cDNA and further amplification and biotin-UTP labeling was performed using TrueLabeling-AMP™ 2.0 cRNA (SABiosciences, Frederick, MD, USA). After purification of labeled target cRNA with the SuperArray ArrayGrade cRNA Cleanup Kit, the cRNA was hybridized to Human Cancer OHS-802 Oligo GEArray membranes that profile 440 genes (both SABiosciences). The expression levels of each gene were detected with chemiluminescence using the alkaline phosphatase-conjugated streptavidin substrate, and membranes were recorded using the MultiImage™ II Light Cabinet (DE-500) (Alpha Innotech Corp., CA, USA).
No related posts.