American Society of Hematology

Figure 5

$Figure 5. Apoptotic mechanism of silvestrol. (A) Silvestrol causes increase in proapoptotic Bcl-2 family members in CLL cells: CLL patient cells were treated with silvestrol (80 nm), cycloheximide (CHX, 100 μM), or flavopiridol (200 nM) for 16 hours. Cells were analyzed by immunoblot for Bmf, Bak, and other Bcl-2 family members. Blot is representative of 4 CLL patient samples tested. Numbers below the lanes show densitometry data for Bmf and Bak levels in treated samples relative to untreated, normalized to the actin control. (B) Silvestrol induces reactive oxygen species (ROS) generation and mitochondrial depolarization in CLL cells: CLL patient cells (n = 3) were treated with silvestrol (80 nm) or flavopiridol (200 nM) for 16 hours. Cells were analyzed by flow cytometry using dihydroethidium (DHE) to detect ROS, JC-1 to detect mitochondrial depolarization, and propidium iodide (PI) to detect cell death. Percentages of live cells (PI negative) are shown as . Percentages of cells showing ROS generation (DHE positive) are shown as and percentages of cells with depolarized mitochondria (monomeric JC-1) are shown as ■. Bars show plus or minus SD. (C) Silvestrol-induced mitochondrial depolarization: CLL patient cells (n = 4) were treated as in panel A, with or without the caspase inhibitor Boc-D-fmk (100 μM). Cells were analyzed at 16 hours by flow cytometry using PI and JC-1. The percentages of cells with intact mitochondria are shown as , and PI-negative cells are shown as ■. Bars show plus or minus SD. Boc-D-fmk efficacy was confirmed by reversal of annexin-FITC positivity in treated cells (not shown). Although Boc-D-fmk reduced mitochondrial depolarization in silvestrol-treated CLL cells, this effect did not reach statistical significance relative to the untreated sample. (D) Silvestrol causes reduction in mitochondrial Smac/DIABLO: CLL patient cells treated with 80 nM silvestrol or 200 nM flavopiridol for 16 hours were subfractionated and analyzed by immunoblot. Voltage-dependent anion channel (VDAC) was used as a control for mitochondrial fractionation.$

Apoptotic mechanism of silvestrol. (A) Silvestrol causes increase in proapoptotic Bcl-2 family members in CLL cells: CLL patient cells were treated with silvestrol (80 nm), cycloheximide (CHX, 100 μM), or flavopiridol (200 nM) for 16 hours. Cells were analyzed by immunoblot for Bmf, Bak, and other Bcl-2 family members. Blot is representative of 4 CLL patient samples tested. Numbers below the lanes show densitometry data for Bmf and Bak levels in treated samples relative to untreated, normalized to the actin control. (B) Silvestrol induces reactive oxygen species (ROS) generation and mitochondrial depolarization in CLL cells: CLL patient cells (n = 3) were treated with silvestrol (80 nm) or flavopiridol (200 nM) for 16 hours. Cells were analyzed by flow cytometry using dihydroethidium (DHE) to detect ROS, JC-1 to detect mitochondrial depolarization, and propidium iodide (PI) to detect cell death. Percentages of live cells (PI negative) are shown as . Percentages of cells showing ROS generation (DHE positive) are shown as and percentages of cells with depolarized mitochondria (monomeric JC-1) are shown as ■. Bars show plus or minus SD. (C) Silvestrol-induced mitochondrial depolarization: CLL patient cells (n = 4) were treated as in panel A, with or without the caspase inhibitor Boc-D-fmk (100 μM). Cells were analyzed at 16 hours by flow cytometry using PI and JC-1. The percentages of cells with intact mitochondria are shown as , and PI-negative cells are shown as ■. Bars show plus or minus SD. Boc-D-fmk efficacy was confirmed by reversal of annexin-FITC positivity in treated cells (not shown). Although Boc-D-fmk reduced mitochondrial depolarization in silvestrol-treated CLL cells, this effect did not reach statistical significance relative to the untreated sample. (D) Silvestrol causes reduction in mitochondrial Smac/DIABLO: CLL patient cells treated with 80 nM silvestrol or 200 nM flavopiridol for 16 hours were subfractionated and analyzed by immunoblot. Voltage-dependent anion channel (VDAC) was used as a control for mitochondrial fractionation.

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