Cgp57380

The human Ph+ ALL cell line SUP-B15 (the American Type Culture Collection, CRL-1929), CML cell line K562 (Hematology Lab, West China Hospital of Sichuan University, Sichuan, China 610041), and primary leukemic blasts were maintained in RPMI 1640 or IMDM (Hyclo, USA), supplemented with 5% penicillin/streptomycin and 10% fetal bovine serum (FBS) in a humidified atmosphere at 37°C in a 5% CO2 incubator. Blast cells were isolated by Ficoll density gradient centrifugation (TBD Science, Tianjin, China). Ribavirin (Sigma, USA) was dissolved in H₂O at a stock concentration of 20mmol/L and sterile filtered, kept at -80°C in small aliquots. Imatinib (Basel, Switzerland), U0126 (Beyotime), and CGP57380 (Sigma) were prepared in dimethyl sulfoxide (DMSO) stored at -20°C with 10 mmol/L and diluted into suitable concentrations before being used.

U2OS cells (purchased from ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D5796) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, E6541L), 2 mM L-Glutamine (LabClinics, M11–004) and 100 μg/ml penicillin-streptomycin (LabClinics, P11-010). For the inhibition of p38α, we used 1 μM PH-797804 (Selleckchem, S2726) or 10 μM SB203580 (Axon MedChem); both compounds can also inhibit p38β, although PH-797804 has been reported to preferentially inhibit p38α^{44 (link)}. MK2 was inhibited using 10 μM MK-2 Inhibitor III (Calbiochem) or 10 μM PF 3644022 (Sigma, PZ0188), whereas MSK activity was inhibited using 5 μM SB 747651 (Axon MedChem), and MNK activity was inhibited using 10 μM CGP-57380 (Sigma). MitoQ (kind gift from M. Murphy, Cambridge, UK) was used at 0.5 μM. Cisplatin was used at 50 μM, and the caspase inhibitors Z-VAD(OMe)-FMK (SM Biochemicals LLC SMFMK001) and Q-VD-OPH (SM Biochemicals LLC SMPH001) at 50 μM. The inhibitors were dissolved in DMSO and the total concentration of DMSO in the culture medium did not exceed 1%.

CGP57380 (C0993) was purchased from Sigma-Aldrich and used at 20 μM. Rapamycin (#9904), LY294002 (#9901), U0126 (#9903) and SB203580 (#5633) were purchased from Cell Signaling Technology (Danvers, MA, USA) and used at 100 nM, 20 μM, 10 μM, and 10 μM respectively. Mouse monoclonal antibodies against NDV phosphoprotein (P protein) and nucleocapsid protein (NP protein) were prepared in our laboratory. Puromycin and mouse monoclonal anti-Puromycin 12D10 (MABE343) were purchased from Merck Millipore (Billerica, MA, USA). Mouse monoclonal anti-FLAG M2 antibody (F1804) and anti-β-actin antibody (A1978) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and other primary antibodies were from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibody, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse and fluorescein (FITC)-conjugated goat anti-rabbit secondary antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Small interfering RNAs (siRNAs) designed specfically for human Mnk1 (sc-39106), eIF4E (sc-35284) and eIF4G (sc-35286) were from Santa Cruz Biotechnology (Dallas, TX, USA). RC DC Protein Assay kit (500–0199) was purchased from Bio-Rad.