Materials and methods Cell lines The T-ALL cell lines, Molt-4 (GC resistant) and Jurkat (GC resistant) were kindly provided by Dr. Stephan W. Morris (St. Jude Children’s Research Hospital). CEM-C1-15 (GC resistant) and CEM-C7-14 (GC sensitive) were kindly provided by Dr. E. Brad Thompson (University of Texas Medical Branch). this website All cell lines were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, St Louis, MO, USA), 2 mM L-glutamine (Gibco),

and antibiotics (penicillin 100 U/mL and streptomycin 50 μg/mL) at 37°C in a humidified 5% CO2 in-air atmosphere. Reagents and antibodies PND-1186 supplier rapamycin (Calbiochem, La Jolla, CA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and used at the concentration of 10 nM. Dex (Sigma) was dissolved in ethanol and used at the concentration of 1 μM. The final concentrations of DMSO and ethanol

in the medium were 0.05% and 0.1%, respectively, at which cell proliferation/growth or viability was not obviously altered. MTT and Propidium iodide (PI) were https://www.selleckchem.com/products/kpt-8602.html purchased from Sigma. Annexin V-PI Kit was purchased from Keygen (Nanjing, China). Antibodies to phospho-4E-BP1, phospho-p70S6K, cyclin D1, p27, Bax, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to p21 was purchased from BD Bioscience (San Jose, CA, USA) Calpain and antibodies to Bim, Mcl-1,

cyclin A, caspase-3 (cleaved at Asp175), NF-κB, and secondary antibodies of horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody and HRP-conjugated sheep anti-mouse antibody were all obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Anti-GAPDH antibody was obtained from Kangchen Bio-Tech (Shanghai, China). Cell treatment Logarithmically growing cells were harvested and replaced in 96- or 6-well sterile plastic culture plates (Corning Inc., Acton, MA, USA), to which 10 nM rapamycin (Rap group), 1 μM Dex (Dex group), 10 nM rapamycin plus 1 μM Dex (Rap+Dex group), and 0.05% DMSO plus 0.1% ethanol (Control group) were added respectively. At the end of the incubation period, cells were transferred to sterile centrifuge tubes, pelleted by centrifugation at 400 g at room temperature for 5 min, and prepared for analysis as described below. Proliferation assay MTT assay is based on the conversion of the yellow tetrazolium salt to purple formazan crystals by metabolically active cells and provides a quantitative estimate of viable cells. Cells were seeded in 96-well plates (20,000/mL) and incubated for 48 h. 0.5 mg/mL MTT (final concentration) was added to each well for 4 h at 37°C. Then, 100% (v/v) of a solubilization solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were re-incubated for 24 h at 37°C.