Inhibition of tyrosine kinase activity of PDGFRα by ponatinib attenuates signaling of β-catenin by lowering its stability. (A) Ponatinib concentration-dependently lowered β-catenin. EOL-1 cells were incubated with ponatinib for 24 h, and cytoplasmic and nuclear extracts were determined by immunoblotting. (B) Analysis of β-catenin localization. EOL-1 cells were pretreated with 1 nM ponatinib for 24 h, immunofluorescence analysis was performed with anti-β-catenin. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) EOL-1 cells were pretreated with the indicated concentrations of ponatinib for 24 h or 1 nM ponatinib for various durations; and the nuclear extracts were then assayed for TCF/LEF activation by EMSA. (D) Ponatinib increased β-catenin turnover rate. After pretreatment with or without 1 nM ponatinib for 16 h, EOL-1 cells were exposed to 5 μg/ml of CHX, followed by immunoblotting for β-catenin. (E) Inhibition of PDGFRα decreased β-catenin. EOL-1 cells were treated with 1 nM ponatinib for various times, then total and tyrosine-phosphorylated β-catenin were evaluated (Left) by immunoblotting. EOL-1 cells were transfected with mock siRNA or PDGFRα siRNA, and β-catenin was monitored by immunoblotting. (F) Ponatinib abrogated TCF/LEF-dependent luciferase activity. EOL-1 cells were transfected with TOPflash and FOPflash plasmids and pEFRenilla-luc. After 24 h, the cells were treated with ponatinib for another 24 h, then underwent luciferase activity assay. (G) Ponatinib decreased the expression of target genes of β-catenin. Immunoblotting analysis in EOL-1 cells that were exposed to ponatinib for 24 h. (H) Ectopically changing the levels of β-catenin affected the ponatinib-mediated apoptosis. Twenty-four hours after transfection with control or Mcl-1 siRNA, or empty vector, pcDNA3-β-catenin, EOL-1 cells were treated with ponatinib, and the relevant protein levels were evaluated by immunoblotting (left); parallel samples were examined by the trypan blue dye exclusion assay (right, *** P<0.0001, t test; error bars represent 95% confidence intervals).