Activation of p-ERK1/2 due to off-target inhibition of DUSP6 by BCI abrogated its antileukemic response. (A) A model depicting JNK1/2 and P38 activation in leukemic cells upon Dusp1 knockdown. (B) Consequently, the expression of proapoptotic proteins BIM and P53 are induced, whereas the expression of antiapoptotic protein BCL2 was significantly reduced. Expression levels were quantified and normalized to the control condition (pMIV-Vector expressing Sc-ShRNA normalized to β-actin). The resulting normalized values are presented below each blot for reference. (C) A model depicting chemical inhibition of DUSP1 and DUSP6 by BCI-activated p-ERK1/2 that suppressed JNK1/2-mediated apoptotic response. (D) Immunoblots showing the activation of p-ERK1/2 upon BCI treatment resulting to inhibition of JNK1/2 and reduced expression of BIM, whereas the levels of P53 and BCL2 were unaffected. Treatment with trametinib alone restored JNK1/2 activation and the level of BIM with modest reduction on BCL2 levels. Interestingly, cells treated with BCI + Tram exhibit induced expression of P53 with reduced BCL2 levels. Representative blots are from 2 independent experiments. Expression levels were assessed and normalized to control condition (vehicle treatment normalized to β-actin). The resulting normalized values are indicated below each blot. Tram, trametinib.