American Society of Hematology

Figure 6

$Figure 6. Role of Bim in Bak and Bax activation and apoptosis mediated by perifosine and PD184352 cotreatment. (A) U937 cells in which Bim was knocked down with shRNA and their control counterpart shGFP-transfected cells were exposed to perifosine (2.5μM) and PD184352 (10μM) for 16 hours, after which cells were lysed in CHAPS buffer and subjected to Bak and Bax conformation analysis, as indicated in “Bax and Bak conformational change.” Alternatively, protein lysates were prepared from cytosolic and membrane fractions, as described in “Subcellular fractionation,” and subjected to Western blot analysis (B). (C) Bim knockdown cells and their control counterparts were treated and lysed as in (A) and analyzed for Bak and Bax binding, as described in Figure 3C. (D) U937 cells were stably transfected with a wild-type Bim construct, and clones exhibiting increases in Bim expression were pooled and used for subsequent experiments (D top panel). Bim-overexpressing cells were exposed to perifosine (2.5μM), PI-103 (3μM), or LY294002 (20μM) for 24 hours, after which the extent of apoptosis was monitored using annexin V analysis assay (D bottom panel). Values represent the means for 3 separate experiments ± SD. *Significantly higher than values for pcDNA3.1-transfected cells (P < .05 in each case). (E) Protein lysates were prepared from 2 clones (shBad-2 and shBad-5) of U937 cells in which Bad was knocked down using shRNAmir, or their control counterparts transfected with EGFP-shRNAmir (shEGFP), after which they were subjected to Western blot analysis (E top panel). These cells were treated with perifosine and PD184352 for 24 hours, after which the extent of cell death was monitored by the annexin V analysis assay (D bottom panel). Values represent the means for 3 separate experiments ± SD. *Not significantly different from values for shEGFP-transfected cells (P > .05 in each case).$

Role of Bim in Bak and Bax activation and apoptosis mediated by perifosine and PD184352 cotreatment. (A) U937 cells in which Bim was knocked down with shRNA and their control counterpart shGFP-transfected cells were exposed to perifosine (2.5μM) and PD184352 (10μM) for 16 hours, after which cells were lysed in CHAPS buffer and subjected to Bak and Bax conformation analysis, as indicated in “Bax and Bak conformational change.” Alternatively, protein lysates were prepared from cytosolic and membrane fractions, as described in “Subcellular fractionation,” and subjected to Western blot analysis (B). (C) Bim knockdown cells and their control counterparts were treated and lysed as in (A) and analyzed for Bak and Bax binding, as described in Figure 3C. (D) U937 cells were stably transfected with a wild-type Bim construct, and clones exhibiting increases in Bim expression were pooled and used for subsequent experiments (D top panel). Bim-overexpressing cells were exposed to perifosine (2.5μM), PI-103 (3μM), or LY294002 (20μM) for 24 hours, after which the extent of apoptosis was monitored using annexin V analysis assay (D bottom panel). Values represent the means for 3 separate experiments ± SD. *Significantly higher than values for pcDNA3.1-transfected cells (P < .05 in each case). (E) Protein lysates were prepared from 2 clones (shBad-2 and shBad-5) of U937 cells in which Bad was knocked down using shRNAmir, or their control counterparts transfected with EGFP-shRNAmir (shEGFP), after which they were subjected to Western blot analysis (E top panel). These cells were treated with perifosine and PD184352 for 24 hours, after which the extent of cell death was monitored by the annexin V analysis assay (D bottom panel). Values represent the means for 3 separate experiments ± SD. *Not significantly different from values for shEGFP-transfected cells (P > .05 in each case).

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