FLT3_ITD627E is sufficient to confer resistance to PKC412 in a reconstitution model. (A) 32D_ITD cells (■) and 32D_ITD627E cells () were treated with increasing concentrations of PKC412 for 24 hours and 48 hours, and the percentage of apoptotic cells was measured as subG₁ DNA content by flow cytometry. (B) Intact mitochondrial outer membrane potentials of 32D_ITD (■) and 32D_ITD627E cells () were assessed by flow cytometric measurement of TMRE-positive cells upon incubation with a range of PKC412 concentrations. Figure 2B illustrates that the FLT3_ITD627E receptor prevents PKC412-induced mitochondrial outer membrane permeabilization. (C) The percentage of apoptotic cells corresponding to subG₁ DNA content was determined by flow cytometry after treatment of 32D_ITD (■), 32D_ITD627E (), and 32D_ITD627A () with PKC412 for 24 hours and 48 hours. In each figure, means of at least 3 independent experiments are depicted. Error bars represent mean (± SD). (D) 32D_ITD (■) and 32D_ITD627E () cells were treated for 48 hours with various concentrations of K252a (left panel) or SU5614 (right panel), and the percentage of apoptotic cells was determined by flow cytometry. Shown are the means of at least 3 independent experiments. Error bars represent mean (± SD).