American Society of Hematology

Figure 5

$Figure 5. Knockdown of Bim, but not Noxa, significantly diminishes sorafenib/obatoclax-mediated cell death. (A) U937 cells were transfected with 2 shRNA constructs designed against Bim (shBim#1 and shBim#2), and one clone from each transfection was selected. These and shGFP control cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) for 48 hours after which Western blot analysis was performed. (B) shBim#1 and shBim#2 cells were treated with sorafenib and obatoclax as in panel A for 6 hours after which the cytosolic fraction was isolated and subjected to Western blot analysis. (C) shBim#1, shBim#2, and shGFP cells were exposed to sorafenib and obatoclax as in panel A for 48 hours after which the extent of cell death was monitored by the 7-AAD staining assay. *Significantly less than values for shGFP control cells; P < .02. (D) U937 cells overexpressing wild-type Bim or their empty vector control (pcDNA3.1) were treated with sorafenib (7.5μM) and obatoclax (1.5μM) for 24 hours after which the percentage of dead cells was determined using the 7-AAD assay. *Significantly greater than values obtained for pcDNA3.1 cells (P < .05). (E) U937 cells in which Noxa was stably knocked down with shRNA and their control counterpart shGFP-transfected cells were exposed to sorafenib and obatoclax for 48 hours after which the extent of cell death was determined using the 7-AAD assay. *Not significantly different from values obtained for shGFP-transfected cells (P > .05). (F) Alternatively, cells were lysed and protein lysates subjected to Western blot analysis to monitor down-regulation of Noxa, and caspase-3 activation by Western blot analysis.$

Knockdown of Bim, but not Noxa, significantly diminishes sorafenib/obatoclax-mediated cell death. (A) U937 cells were transfected with 2 shRNA constructs designed against Bim (shBim#1 and shBim#2), and one clone from each transfection was selected. These and shGFP control cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) for 48 hours after which Western blot analysis was performed. (B) shBim#1 and shBim#2 cells were treated with sorafenib and obatoclax as in panel A for 6 hours after which the cytosolic fraction was isolated and subjected to Western blot analysis. (C) shBim#1, shBim#2, and shGFP cells were exposed to sorafenib and obatoclax as in panel A for 48 hours after which the extent of cell death was monitored by the 7-AAD staining assay. *Significantly less than values for shGFP control cells; P < .02. (D) U937 cells overexpressing wild-type Bim or their empty vector control (pcDNA3.1) were treated with sorafenib (7.5μM) and obatoclax (1.5μM) for 24 hours after which the percentage of dead cells was determined using the 7-AAD assay. *Significantly greater than values obtained for pcDNA3.1 cells (P < .05). (E) U937 cells in which Noxa was stably knocked down with shRNA and their control counterpart shGFP-transfected cells were exposed to sorafenib and obatoclax for 48 hours after which the extent of cell death was determined using the 7-AAD assay. *Not significantly different from values obtained for shGFP-transfected cells (P > .05). (F) Alternatively, cells were lysed and protein lysates subjected to Western blot analysis to monitor down-regulation of Noxa, and caspase-3 activation by Western blot analysis.

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