Ponatinib mediates caspase-3-dependent cleavage of Mcl-1. (A) Ponatinib precipitated in Mcl-1 turnover. After pretreatment with or without 1 nM ponatinib, EOL-1 cells were exposed to 5 μg/ml of cycloheximide (CHX), followed by Mcl-1 detection with immunoblotting. (B) MG-132 did not abrogate ponatinib-induced cleavage of Mcl-1. EOL-1 cells were treated with 1 nM ponatinib in the presence or absence of 0.5 μM MG-132 for 24 h. Mcl-1 level was then monitored with immunoblotting. (C) Mcl-1 cleavage occurred with onset of apoptosis after treatment with ponatinib. EOL-1 cells were treated with 1 nM ponatinib for different times, and the indicated proteins were measured with immunoblotting. (D) Mcl-1 cleaved in a caspase-3-dependent manner. EOL-1 cells were treated with 1 nM ponatinib for 24 h with or without 10 μM z-DEVD-fmk, then underwent immunoblotting. (E) Silencing Mcl-1 potentiated ponatinib-induced apoptosis in EOL-1 cells. Twenty-four hours after transfection with Mcl-1 siRNA or control (mock) siRNA, EOL-1 cells were treated with various concentrations of ponatinib, and levels of Mcl-1, PARP, and actin were evaluated by immunoblotting (top); parallel samples were examined for apoptosis by trypan blue staining (bottom, *** P < 0.0001, t test, error bars represent 95% confidence intervals; representative data from 3 independent experiments are shown). (F) Enforced overexpression of Mcl-1 abrogated the ponatinib-induced apoptosis. Twenty-four hours after transfection with pCMV5-flag empty vector or the plasmid expressing Mcl-1, EOL-1 cells were incubated with or without concentrations of ponatinib for another 24 h. Cell viability was evaluated by trypan blue dye exclusion (lower, *** P < 0.0001, t test, error bars represent 95% confidence intervals); Mcl-1 and PARP levels were detected by immunoblotting.