Figure 4.
Figure 4. Imatinib inhibits the capacity to induce CLL-cell expression of Bid and CD95 except in CLL cells made to express imatinib-resistant mutant C-Ab. (A) CLL cells were transduced with an adenovirus encoding TAp73α (Ad-TAp73α) or lacking a transgene (Ad-blank), as indicated at the bottom of the panel. At 24 hours after the start of transduction, the CLL cells were isolated and cultured in fresh medium for another 48 hours. Cell lysates were prepared for immunoblot analyses using antibodies specific for p73 (TAp73α), CD95, Bid, or β-actin, as indicated on the right of each immunoblot. (B) CLL cells were transduced with an adenovirus expressing p73 (Ad-TAp73α) or with an adenovirus lacking transgene expression (Ad-blank) in the presence or absence of imatinib at 10 μM. After 48 hours the CLL cells were washed and cultured in fresh medium for another 24 hours and then examined for expression of surface CD95 by flow cytometry. Representative histograms show the expression of CD95 of Ad-blank- or Ad-TAp73α-transduced CLL cells cultured without (filled gray) or with (thick line) imatinib, as indicated at the top of each histogram. The thin lined open histogram corresponds to that of transduced CLL cells cultured without imatinib and stained with an isotype control mAb of irrelevant specificity. (C) CLL cells were transduced with an adenovirus encoding the Flag-tagged, imatinib-resistant c-Abl (Ad-c-Abl (T315I)) or Ad-blank, as indicated at the top of the immunoblot. Twenty-four hours later the CLL cells were washed extensively and then cocultured with CD32-expressing mouse L-cells and an isotype control mAb (control) or the agonist anti-CD40 mAb (α-CD40), in media containing (+)or lacking (-) imatinib at 10 μM, as indicated at the bottom of the panel. Twenty-four hours later, the CLL cells were isolated and used to prepare cell lysates. Fifty μg of each lysate was loaded onto separate lanes of a PAGE gel for immunoblot analyses. Membranes were probed with antibodies specific for p73, Bid, the Flag tag (c-Abl (α-Flag)), or β-actin, as indicated to the right of each blot.

Imatinib inhibits the capacity to induce CLL-cell expression of Bid and CD95 except in CLL cells made to express imatinib-resistant mutant C-Ab. (A) CLL cells were transduced with an adenovirus encoding TAp73α (Ad-TAp73α) or lacking a transgene (Ad-blank), as indicated at the bottom of the panel. At 24 hours after the start of transduction, the CLL cells were isolated and cultured in fresh medium for another 48 hours. Cell lysates were prepared for immunoblot analyses using antibodies specific for p73 (TAp73α), CD95, Bid, or β-actin, as indicated on the right of each immunoblot. (B) CLL cells were transduced with an adenovirus expressing p73 (Ad-TAp73α) or with an adenovirus lacking transgene expression (Ad-blank) in the presence or absence of imatinib at 10 μM. After 48 hours the CLL cells were washed and cultured in fresh medium for another 24 hours and then examined for expression of surface CD95 by flow cytometry. Representative histograms show the expression of CD95 of Ad-blank- or Ad-TAp73α-transduced CLL cells cultured without (filled gray) or with (thick line) imatinib, as indicated at the top of each histogram. The thin lined open histogram corresponds to that of transduced CLL cells cultured without imatinib and stained with an isotype control mAb of irrelevant specificity. (C) CLL cells were transduced with an adenovirus encoding the Flag-tagged, imatinib-resistant c-Abl (Ad-c-Abl (T315I)) or Ad-blank, as indicated at the top of the immunoblot. Twenty-four hours later the CLL cells were washed extensively and then cocultured with CD32-expressing mouse L-cells and an isotype control mAb (control) or the agonist anti-CD40 mAb (α-CD40), in media containing (+)or lacking (-) imatinib at 10 μM, as indicated at the bottom of the panel. Twenty-four hours later, the CLL cells were isolated and used to prepare cell lysates. Fifty μg of each lysate was loaded onto separate lanes of a PAGE gel for immunoblot analyses. Membranes were probed with antibodies specific for p73, Bid, the Flag tag (c-Abl (α-Flag)), or β-actin, as indicated to the right of each blot.

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