Dose-dependent effects of ponatinib on endothelial cell ROS production and viability. (A) ROS generation by SVEC4-10 endothelial cells in culture measured by mean (± SEM) H2DCFDA fluorescence after 24 hours of ponatinib treatment. Positive control data are shown for phorbol 12-myristate 13-acetate (PMA)–treated cells. (B) Examples of SVEC4-10 cells exposed for 24 hours to ponatinib (0.1 to 2.5 μM) by fluorescent imaging of H2DCFDA and DAPI (nuclear staining) illustrating dose-dependent increase in ROS, and low-magnification bright-field (LM-BF) microscopy illustrating loss of cell confluence at high concentrations. (C) Mean (± SEM) cell viability after 24 hours of exposure to ponatinib (0.1 to 2.5 μM) determined by the proportion of cells staining positive for PI. *P < .05 vs all other conditions by post hoc testing after correction for multiple comparisons. (D) Cell viability at various concentrations of ponatinib (Pon) with and without NAC. *P < .05. (E-F) Mean (± SEM) area for externalized VWF on HUVECs in a microfluidic chamber after 24 hours of exposure to ponatinib (0.5 μM), TNF-α (10 ng/mL), or control serum, and examples of fluorescent microscopy for each condition. *P < .01. AU, arbitrary unit.