Fig. 1

Growth inhibition and cellular signaling following axitinib/ponatinib treatment in T315I-mutant and compound-mutant cells. a Ba/F3 T315I or Ba/F3 ponatinib-resistant (Ba/F3 ponatinib-R) cells were exposed to axitinib or ponatinib for 72 h at the indicated concentrations and subjected to quantitative cell proliferation analysis. Each result is presented as the mean percentage of proliferation relative to unexposed control cultures. *P < 0.05 compared to T315I cells. b Ba/F3 T315I or Ba/F3 ponatinib-R cells were treated with ponatinib (10 nM) or axitinib (500 nM) for 24 h. Total cell extracts were examined via immunoblot analysis with anti-phospho ABL, phospho-Crk-L, phospho-S6, cleaved caspase 3, cleaved PARP, ABL, Crk-L, and β-actin antibodies. c Caspase activity was analyzed using an ApoAlert® Caspase-3 Colorimetric Assay Kit (Takara Bio Inc. Otsu, Shiga, Japan) according to the manufacturer’s protocol. Data represent three independent sets of experiments. *P < 0.05 compared to Ba/F3 T315I and Ba/F3 ponatinib-R cells. d Apoptosis in Ph-positive cell lines was assayed using a FITC Annexin V Apoptosis Detection Kit I™ (BD Pharmingen, San Jose, CA, USA). The experiments were performed in triplicate. e Ba/F3 T315I or Ba/F3 ponatinib-R cells were treated with axitinib at the indicated concentrations for 24 h. Total cell extracts were examined via immunoblot analysis with anti-phospho ABL, phospho-Crk-L, phospho-S6, cleaved caspase 3, cleaved PARP, ABL, Crk-L, and β-actin antibodies. f Akt activity was analyzed using a phospho-AKT 1/2/3 (Ser473) InstantOne™ enzyme-linked immunosorbent assay kit (Affymetrix, Cleveland, OH, USA) according to the manufacturer’s protocol. Data represent three independent sets of experiments. *P < 0.05 compared to Ba/F3 T315I and Ba/F3 ponatinib-R cells. g, h T315I-positive or compound-mutant primary cells were subjected to quantitative cell proliferation analysis after a 72-h exposure to axitinib or ponatinib. Each result is presented as the mean percentage of proliferation relative to unexposed control cultures. *P < 0.05 compared to control. i T315I-positive or compound-mutant primary cells were treated with axitinib at the indicated concentrations for 24 h. Total cell extracts were examined via immunoblot analysis with anti-phospho-Crk-L, phospho-S6, cleaved PARP, and β-actin antibodies