Figure 6
Ectopic expression of Bcl-xL or Bcl-2 blocks PD184352/UCN-01–induced apoptosis without affecting phosphorylation status of BimEL. (A) RPMI8226 cells were stably transfected with human wild-type Bcl-xL construct or its empty vector (pSSFV). The stable clones (1 for empty-vector control and 2 for Bcl-xL designated w1 and w8) were obtained by selection with G418, and expression of Bcl-xL was monitored by Western blot analysis (inset). 8226/Bcl-xL and 8226/pSFFV cells were exposed to 150 nM UCN-01 and 5 μM PD184352 or 20 μM U0126 for 24 hours, after which the percentage of dead cells (7-AAD+) was determined by 7-AAD staining and flow cytometry. (B) U266 cells were stably transfected with wild-type mouse Bcl-2 (moBcl-2) and its empty vector (pUSE). The stable clones (1 for pUSE and 2 for moBcl-2 designated 1B3 and 1B4) were obtained by selection with G814, and Western blot analysis was performed to monitor expression of either mouse Bcl-2 or endogenous Bcl-2 using antibodies against mouse and human Bcl-2 (inset), respectively. These cells were exposed to 100 nM UCN-01 plus or minus 5 μM PD184352 for 40 hours, and the percentage of dead cells (7-AAD+) was determined by flow cytometry. Results represent the means (± SD) for 3 separate experiments performed in triplicate. **Significantly lower than the value for empty-vector controls with the same treatment (P < .001). (C,D) 8226/Bcl-xL (clone w1), U266/Bcl-2 (clone 1B4), and their empty-vector control cells were treated as described for panels A and B, after which WB was performed to assess expression of phospho-ERK1/2, Bim, cleavage of caspase-3, and PARP degradation. Phosphorylated forms of BimEL are manifested by slowly migrating species (arrows). CF indicates cleavage fragments. Alternatively, for 8226/Bcl-xL and 8226/pSFFV cells in Figure 6C, cytosolic S-100 fractions were prepared and subjected to WB to monitor cytochrome c expression as described in “Materials and methods.” Each lane was loaded with 20 μg protein, and blots were stripped and reprobed with antiactin or antitubulin antibodies to ensure equal loading and transfer. Two additional studies yielded equivalent results.

Ectopic expression of Bcl-xL or Bcl-2 blocks PD184352/UCN-01–induced apoptosis without affecting phosphorylation status of BimEL. (A) RPMI8226 cells were stably transfected with human wild-type Bcl-xL construct or its empty vector (pSSFV). The stable clones (1 for empty-vector control and 2 for Bcl-xL designated w1 and w8) were obtained by selection with G418, and expression of Bcl-xL was monitored by Western blot analysis (inset). 8226/Bcl-xL and 8226/pSFFV cells were exposed to 150 nM UCN-01 and 5 μM PD184352 or 20 μM U0126 for 24 hours, after which the percentage of dead cells (7-AAD+) was determined by 7-AAD staining and flow cytometry. (B) U266 cells were stably transfected with wild-type mouse Bcl-2 (moBcl-2) and its empty vector (pUSE). The stable clones (1 for pUSE and 2 for moBcl-2 designated 1B3 and 1B4) were obtained by selection with G814, and Western blot analysis was performed to monitor expression of either mouse Bcl-2 or endogenous Bcl-2 using antibodies against mouse and human Bcl-2 (inset), respectively. These cells were exposed to 100 nM UCN-01 plus or minus 5 μM PD184352 for 40 hours, and the percentage of dead cells (7-AAD+) was determined by flow cytometry. Results represent the means (± SD) for 3 separate experiments performed in triplicate. **Significantly lower than the value for empty-vector controls with the same treatment (P < .001). (C,D) 8226/Bcl-xL (clone w1), U266/Bcl-2 (clone 1B4), and their empty-vector control cells were treated as described for panels A and B, after which WB was performed to assess expression of phospho-ERK1/2, Bim, cleavage of caspase-3, and PARP degradation. Phosphorylated forms of BimEL are manifested by slowly migrating species (arrows). CF indicates cleavage fragments. Alternatively, for 8226/Bcl-xL and 8226/pSFFV cells in Figure 6C, cytosolic S-100 fractions were prepared and subjected to WB to monitor cytochrome c expression as described in “Materials and methods.” Each lane was loaded with 20 μg protein, and blots were stripped and reprobed with antiactin or antitubulin antibodies to ensure equal loading and transfer. Two additional studies yielded equivalent results.

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