Combined exposure to UCN-01 and L744832 inactivates Akt signaling pathways independently of mitochondrial-mediated apoptosis. (A) U937 and Jurkat cells were treated with 10 μM L744832 with or without UCN-01 (U937, 100 nM; Jurkat, 150 nM) for 18 hours, after which Western blot analysis was performed to evaluate expression of total and/or phosphorylated Akt and its downstream targets (eg, GSK-3α/β, FKHR/FKHRL, p70/p85 S6 kinase, mTOR, 4E-BP1, Bad, and caspase-9). (B) Phosphorylation of Akt, GSK-3α/β, Bad, and caspase-9 was assessed in HL-60 and Raji cells after treatment with 10 μM L744832 ± UCN-01 (HL-60, 100 nM; Raji, 150 nM) for 18 hours. (C) Bcl-2/U937 and pCEP/U937 (left panels) or Bcl-xL/U937 and pcDNA3.1/U937 cells (right panels) were exposed to 10 μM L744832 (L) + 100 nM UCN-01 (U) for 18 hours, after which the phosphorylation status of Akt, GSK-3α/β, FKHR/FKHRL, p70/p85 S6 kinase, 4E-BP1, Bad, and caspase-9 was monitored by Western blot analysis. In each panel, 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.