American Society of Hematology

Figure 4.

$Figure 4. LY294002-induced activation of the mitochondrial apoptotic pathway in DLBCL cell lines. (A) LY294002-induced activation of caspase-8 and cleavage of BID. SUDHL-4, SUDHL-5, SUDHL-8, and OCI-LY19 cells were treated with 10 and 25 μM LY294002 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against caspase-8, BID, and actin as indicated. (B) Loss of mitochondrial membrane potential by LY294002 treatment in DLBCL cells. SUDHL-4, SUDHL-5, SUDHL-8, SUDHL10, and OCI-LY19 cells were treated with and without 25 μM LY294002 for 24 hours. Live cells with intact mitochondrial membrane potential (▪) and dead cells with lost mitochondrial membrane potential (▦) were measured by JC-1 staining and analyzed by flow cytometry as described in “Patients, materials, and methods.” (C) LY294002-induced release of cytochrome c. SUDHL-4, SUDHL-5, and OCI-LY19 cell lines were treated with and without 25 μM LY294002 for 24 hours. Mitochondrial-free cytoplasmic as well as mitochondrial fractions were isolated as described in “Patients, materials, and methods.” Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cytochrome c and actin as indicated.$

LY294002-induced activation of the mitochondrial apoptotic pathway in DLBCL cell lines. (A) LY294002-induced activation of caspase-8 and cleavage of BID. SUDHL-4, SUDHL-5, SUDHL-8, and OCI-LY19 cells were treated with 10 and 25 μM LY294002 for 24 hours. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against caspase-8, BID, and actin as indicated. (B) Loss of mitochondrial membrane potential by LY294002 treatment in DLBCL cells. SUDHL-4, SUDHL-5, SUDHL-8, SUDHL10, and OCI-LY19 cells were treated with and without 25 μM LY294002 for 24 hours. Live cells with intact mitochondrial membrane potential (▪) and dead cells with lost mitochondrial membrane potential (▦) were measured by JC-1 staining and analyzed by flow cytometry as described in “Patients, materials, and methods.” (C) LY294002-induced release of cytochrome c. SUDHL-4, SUDHL-5, and OCI-LY19 cell lines were treated with and without 25 μM LY294002 for 24 hours. Mitochondrial-free cytoplasmic as well as mitochondrial fractions were isolated as described in “Patients, materials, and methods.” Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cytochrome c and actin as indicated.

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