Sorafenib/obatoclax-mediated lethality involves Mcl-1 down-regulation. (A) U937 cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for 6 or 24 hours after which protein lysates were prepared and subjected to Western blot analysis using the designated Abs. (B) HL-60, MV4-11 cells, and primary blasts were treated with sorafenib (7.5μM for HL-60 cells and primary blasts; 75nM for MV4-11cells) and obatoclax (2μM for HL-60, and 0.5μM for MV4-11 and primary blasts) for 28 hours after which cells were lysed and protein lysates were subjected to Western blot analysis. (C) U937 cells ectopically expressing Mcl-1 or their empty vector control cells (pCEP4) were treated with the designated concentrations of sorafenib and obatoclax alone or in combination for 48 hours after which the extent of cell death was determined using the 7-AAD staining assay. Values represent the means for 3 separate experiments ± SD. *Significantly less than values for pCEP4 control cells; P < .05. **P < .01. (D) Alternatively, cleavage of PARP and caspase-3 in U937/Mcl-1 cells exposed to 12μM sorafenib and 3μM obatoclax was monitored by Western blot analysis. (E) U937 cells were treated with sorafenib (7.5μM) and obatoclax (1.5μM) individually or together for 8 or 24 hours after which cells were lysed and subjected to immunoprecipitation using Bim Abs. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with either Bcl-2 (top panel) or Bcl-xL (bottom panel) Abs.