Effects of OM and ponatinib on BCR-ABL-expressing point mutant cells. (A) Ba/F3 T315I cells were cultured at a density of 5 × 10⁶ cells with 50 μg/mL N-ethyl-N-nitrosourea for 24 hours and washed 3 times with RPMI medium. The cells were cultured in RPMI medium with ponatinib, and resistant clones were identified (Ba/F3 ponatinib-resistant [Ba/F3 ponatinib-R]). These Ba/F3 ponatinib-R cells were treated with OM for 72 hours. The number of viable cells was calculated for each group. Results from 3 independent experiments were averaged. *P < .05, OM treatment vs control. (B) Ba/F3 ponatinib-R cells were treated with ponatinib at the indicated concentrations for 72 hours. The number of viable cells was calculated for each group. Results from 3 independent experiments were averaged. (C) The percentage of apoptotic cells was estimated using an annexin V assay after culturing the cells for 48 hours with OM. Results from 3 independent experiments were averaged. Error bars represent standard deviations. *P < .05, OM treatment vs control in the same cell line. (D) Ba/F3 ponatinib-R cells were treated with OM or ponatinib for 48 hours. Total extracts were analyzed by immunoblotting with phospho-specific anti-Abl, Crk-L, cleaved caspase 3, cleaved PARP, Abl, Crk-L, HSP90, Bcl-2, and c-Myc antibodies. Actin was used as the loading control. Data are representative of 3 separate experiments. (E) Ponatinib-resistant Ph-positive primary cells were cultured at a density of 4 × 10⁵ cells/well in the presence of ponatinib and OM for 72 hours. The number of viable cells was calculated for each group. Results from 3 independent experiments were averaged. *P < .05, OM treatment vs control in the same cell line.