Ponatinib

Approximately 15 × 10⁶ cells were grown and exposed to different drugs to examine the regulation of creatine uptake. Fewer cells, approximately 2 × 10⁶, were used in experiments for immunoblot analyses. Drug dose-response experiments were initially performed with different compounds to determine the effective concentrations. For ouabain, cells were incubated in increasing concentrations, starting at 1.0 nM and increased by order of 10 until the concentration of 10 μM was achieved. Separate populations of the cells were also treated with dasatinib or ponatinib (Selleckchem), dual Bcr-Abl and Src family tyrosine kinase inhibitors. The creatine competitive inhibitor, 3-Guanidinopropionic Acid (Sigma-Aldrich, St. Louis, MO), was reconstituted in media (above) and a 30-mM concentration used to treat Myl-R cells. Cells were treated for 24 hours before metabolite extraction. The drugs/compounds were reconstituted in incubation media (3-Guanidinopropionic Acid), hot, autoclaved water (ouabain), and DMSO (imatinib, ponatinib, dasatinib); likewise incubation media, diH₂O, and DMSO were also used as the vehicle controls.

Ba/F3 cells expressing Bcr-Abl^WT and Bcr-Abl^T315I were obtained from Dr. Shah’s laboratory (UCSF, San Francisco, CA, USA). The cells were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (v/v, HyClone, ThermoScientific) and 1× penicillin, strepatmycin, and glutamine (GE Healthcare) at 37 °C and 5.0% CO₂. Imatinib and ponatinib were purchased from Selleckchem. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from Cell Signaling (Boston, MA, USA): anti-Abl, anti-phospho-Abl (Y412), anti-phospho-STAT5 (Tyr694), and anti-STAT5. Anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), whereas anti-rabbit secondary antibody was obtained from Abcam (Cambridge).