Inducible expression of myristoylated Akt but not constitutively active MEK attenuates the lethality of UCN-01/L744832 regimen in human leukemia cells. (A) Jurkat cells stably transfected with a Tet-on inducible constitutively active MEK construct were exposed to 10 μM L744832 (L) + 150 nM UCN-01 (U) for 8 hours following a 24-hour induction with 2 μg/mL doxycycline (Dox), after which the percentage of apoptotic cells was determined by annexin V-FITC/flow cytometry. (B) MEK/Tet-on Jurkat cells were treated as in panel A, after which cells were lysed and subjected to Western blot analysis to monitor expression of MEK, phosphorylation of ERK1/2 and JNK1, and cleavage of PARP. (C) Jurkat cells (clone no. 2 and clone no. 29) stably transfected with a Tet-on inducible myristoylated Akt construct were incubated for 8 hours with 10 μM L744832 + 150 nM UCN-01 following a 24-hour induction with 2 μg/mL Dox, after which annexin V-FITC/flow cytometry was performed to determine the percentage of apoptotic cells. (D) Alternatively, 2 clones (no. 2 and no. 29) of Akt/Tet-on Jurkat cells were treated as described in panel C, after which Western blot analysis was employed to assess expression of myr-Akt, phosphorylation status of mTOR, Bad, and JNK1, and cleavage of PARP. For panels A and C, the results represent the means ± SD for 3 separate experiments performed in triplicate. ^***P < .02 and ^**P < .05); significantly lower than values for cells of the same clone exposed to L744832 + UCN-01 without Dox-induction. For panels B and D, 30 μg protein was loaded in each lane; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. The results were representative of 3 separate experiments. CF indicates cleavage fragment. Two additional studies yielded equivalent results.