Figure 3

Ponatinib induces apoptosis in FIP1LI-PDGFRα-expressing cells. (A) EOL-1 and BaF3-WT or -T674I FIP1L1-PDGFRα cells were exposed to increasing concentrations of ponatinib for 24 h, apoptotic cells were assayed with flow cytometry by PI/Annexin V-FITC (EOL-1) or 7-AAD/Annexin V-PE (BaF3-WT or -T674I FIP1L1-PDGFRα cells) staining. Left, representative histograms; Right, statistical data of 3 independent experiments, the vertical axis stands for the sum of all dead cells. Error bars represent 95% confidence intervals. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) The indicated cells were treated with or without ponatinib (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells, respectively) for 24 h, washed with PBS and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Representative photographs (9700×) were acquired under transmission electron microscopy. (C) The concentration- (for 24 h) and time-dependent (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells) cleavage of PARP and caspase-3 triggered by ponatinib was analyzed by immunoblotting. (D) Ponatinib elicited release of AIF and cytochrome c into the cytosol. Cells were treated with 1 nM ponatinib for the indicated durations and the cytosolic fraction was extracted with digitonin buffer. Levels of AIF and Cytochrome c (Cyto c) were detected by immunoblotting. (E) Immunoblotting of apoptosis-related proteins in CEL cells after treatment for 24 h.