L744832/UCN-01-induced apoptosis is primarily mediated by activation of the intrinsic, mitochondrial pathway. (A) U937 cells were exposed to 10 μM L744832 in either the presence or absence of 100 nM UCN-01 for 18 hours, after which expression of Bcl-2 family molecules (eg, Bcl-2, Bcl-xL, Bax, Bak, and Mcl-1) and XIAP was evaluated by Western blot analysis. (B) U937 cells stably transfected with Bcl-2 or its empty vector (pCEP) or, alternatively, Bcl-xL, dominant-negative (DN) caspase-8 (C8), or their respective empty vector (pcDNA3.1) were treated with 10 μM L744832 (L) + 100 nM UCN-01 (U) for 18 hours, after which the percentage of apoptotic cells was determined by annexin V-FITC/flow cytometry. Results represent the means ± SD for 3 separate experiments performed in triplicate. (C-D) Bcl-2/U937, pCEP/U937, Bcl-xL/U937 and pcDNA3.1/U937 cells were treated as described in panel B, after which whole cells were lysed and subjected to Western blot analysis to monitor expression of Bcl-2 or Bcl-xL and cleavage of caspases, Bid, PARP (C), Mcl-1, and XIAP (D). Alternatively, S-100 fractions were prepared as described in “Materials and methods,” and Western blot analysis was performed to assess cytosolic distribution of cytochrome c, Smac/DIABLO, and AIF. (E) DN caspase-8/U937 and pcDNA3.1/U937 cells were treated as described in panel B, after which Western blot analysis was employed to monitor expression of caspase-8 and cleavage of caspases, Bid, and PARP. For panels A and C-E, each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed for expression of β-actin, as indicated, to ensure equivalent loading and transfer of protein. Two additional studies yielded equivalent results. CF indicates cleavage fragment.