Enforced MEK/ERK activation requires ectopic expression of Akt to protect cells from UCN-01/L744832-mediated apoptosis, while JNK activation plays a functional role in the lethality of this regimen. (A) U937 cells were stably transfected with constitutively active myr-Akt (Akt/U937) and its empty vector (Neo/U937), and Western blot analysis was performed to monitor expression of Akt. (B) Akt/U937 and Neo/U937 cells were transiently transfected with either MEK/EGFP or enhanced GFP (EGFP). After 24 hours, flow cytometry was employed to monitor transfection efficiency by determining the percentage of GFP-positive cells within the gated areas. Results are representative of 3 separate experiments. (C) MEK/EGFP and EGFP-transfected Akt/U937 or Neo/U937 cells, after a 24-hour recovery, were incubated for 8 hours with 10 μM L744832 + 100 nM UCN-01, after which the percentage of viable cells was determined using the Guava ViaCount assay as described in “Materials and methods.” (D) Alternatively, cells were stained by annexin V-PE and subjected to flow cytometric analysis to determine the percentage of annexin V-positive/GFP-positive cells. (E) U937 cells were transfected with JNK1 siRNA oligonucleotides as described in “Materials and methods.” After 24 hours, the levels of JNK1 and JNK2 were monitored by Western blot analysis. (F) Following a 24-hour recovery, JNK1 siRNA-transfected U937 cells were exposed to 10 μM L744832 + 100 nM UCN-01 for 8 hours, after which the percentage of viable cells was determined by ViaCount assay. For panels A and E, each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. Two additional studies yielded equivalent results. For panels C, D, and F, values represent the means ± SD for 3 separate experiments performed in triplicate.