Perifosine/PD184352-mediated lethality involves Mcl-1 down-regulation. (A) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5μM) alone or in combination for 6 or 16 hours, after which protein lysates were prepared and subjected to Western blot analysis using designated antibodies (CF = cleavage fragment). (B) U937 cells were pretreated with zVAD-fmk (25μM) for 1 hour before treatment with perifosine and PD184352 for 16 hours. Then Mcl-1 protein levels and caspase-3 cleavage were monitored by Western blot analysis. (C) Two clones (Mcl1-14 and Mcl1-16) of U937 cells overexpressing Mcl-1 and empty-vector cells (pCEP4) were treated with perifosine and PD184352 for 16 hours, after which Mcl-1 protein levels were monitored by Western blot analysis. Alternatively, the extent of apoptosis was monitored at 24 hours after treatment using annexin V staining assay (D). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for empty-vector pCEP4 cells (P < .005).