American Society of Hematology

Fig. 7.

$Fig. 7. Effect of cotreatment of CLL cells with the PP2Ac inhibitor okadaic acid. / Cotreatment of CLL cells with the PP2Ac inhibitor okadaic acid blocks rolipram/forskolin-induced mitochondrial depolarization and accumulation of cytoplasmic cytochrome c. (A) CLL cells were cultured for 18 hours in media (CT), 5 nM okadaic acid (OA), 10 μM rolipram and 40 μM forskolin (R/F), or the combination of okadaic acid, rolipram, and forskolin (R/F + OA). Mitochondrial depolarization was assessed by means of DiOC6(3) flow cytometry. The percentage of cells with depolarized mitochondria (reduced fluorescence) is shown. Similar results were obtained in 4 of 5 CLL patients. (B) CLL cells cultured as for panel A were subfractionated into heavy-membrane and cytosolic fractions, and immunoblotted for cytochrome c. Total cytochrome c, shown at the bottom, represents a sample in which equivalent portions of cytosolic and membrane fractions were combined. (C) CLL cells cultured as for panel A were immunoprecipitated with a BAD-specific antibody, then immunoblotted for phospho-Ser112 (top), 14-3-3 (second row), or BAD (third row). In addition, whole-cell lysates from the same samples were immunoblotted to assess total BAD (bottom). Similar results were obtained in leukemic cells from 3 of 5 CLL patients so examined.$

Effect of cotreatment of CLL cells with the PP2Ac inhibitor okadaic acid.

Cotreatment of CLL cells with the PP2Ac inhibitor okadaic acid blocks rolipram/forskolin-induced mitochondrial depolarization and accumulation of cytoplasmic cytochrome c. (A) CLL cells were cultured for 18 hours in media (CT), 5 nM okadaic acid (OA), 10 μM rolipram and 40 μM forskolin (R/F), or the combination of okadaic acid, rolipram, and forskolin (R/F + OA). Mitochondrial depolarization was assessed by means of DiOC₆(3) flow cytometry. The percentage of cells with depolarized mitochondria (reduced fluorescence) is shown. Similar results were obtained in 4 of 5 CLL patients. (B) CLL cells cultured as for panel A were subfractionated into heavy-membrane and cytosolic fractions, and immunoblotted for cytochrome c. Total cytochrome c, shown at the bottom, represents a sample in which equivalent portions of cytosolic and membrane fractions were combined. (C) CLL cells cultured as for panel A were immunoprecipitated with a BAD-specific antibody, then immunoblotted for phospho-Ser112 (top), 14-3-3 (second row), or BAD (third row). In addition, whole-cell lysates from the same samples were immunoblotted to assess total BAD (bottom). Similar results were obtained in leukemic cells from 3 of 5 CLL patients so examined.

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