Figure 5
Figure 5. Knockdown of Bim significantly diminishes perifosine/PD184352-mediated cell death. (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. (B) Primary AML mononuclear cells isolated from 3 patients were treated with perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor Bim and Mcl-1 protein levels. (C left panel) Jurkat cells (MT6) inducibly expressing constitutively active MEK1 were left untreated or treated for 48 hours with 2 μg/mL doxycycline, after which cells were analyzed for phospho-ERK1/2 and Bim expression by Western blot. Alternatively, cells were treated with perifosine (8μM) or LY294002 (50μM) for 48 hours, after which cell death was assessed by annexin V analysis (C right panel). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained in the absence of doxycycline (P < .01). (D) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5 μM) alone or in combination for 16 hours, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation assay using Bim antibodies. The immunoprecipitates were then subjected to immunoblot analysis with either Mcl-1 or Bim antibodies. Input lysates were also subjected to Western blot analysis to monitor Bim levels. (E) Two clones (shBim-8 and shBim-13) of U937 cells in which Bim was knocked down using shRNA and their control counterpart shGFP-transfected cells were treated with perifosine and PD184352 alone or in combination for 24 hours, after which the extent of apoptosis was determined using the annexin V staining assay. Alternatively, protein lysates were prepared and Western blot was performed (F). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for shGFP-transfected cells; P < .01. (C inset) Western blot performed on lysates prepared from these cells before treatment.

Knockdown of Bim significantly diminishes perifosine/PD184352-mediated cell death. (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. (B) Primary AML mononuclear cells isolated from 3 patients were treated with perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor Bim and Mcl-1 protein levels. (C left panel) Jurkat cells (MT6) inducibly expressing constitutively active MEK1 were left untreated or treated for 48 hours with 2 μg/mL doxycycline, after which cells were analyzed for phospho-ERK1/2 and Bim expression by Western blot. Alternatively, cells were treated with perifosine (8μM) or LY294002 (50μM) for 48 hours, after which cell death was assessed by annexin V analysis (C right panel). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained in the absence of doxycycline (P < .01). (D) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5 μM) alone or in combination for 16 hours, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation assay using Bim antibodies. The immunoprecipitates were then subjected to immunoblot analysis with either Mcl-1 or Bim antibodies. Input lysates were also subjected to Western blot analysis to monitor Bim levels. (E) Two clones (shBim-8 and shBim-13) of U937 cells in which Bim was knocked down using shRNA and their control counterpart shGFP-transfected cells were treated with perifosine and PD184352 alone or in combination for 24 hours, after which the extent of apoptosis was determined using the annexin V staining assay. Alternatively, protein lysates were prepared and Western blot was performed (F). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for shGFP-transfected cells; P < .01. (C inset) Western blot performed on lysates prepared from these cells before treatment.

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