American Society of Hematology

Figure 3.

$Figure 3. Activation of multiple caspases by brefeldin A. (A) Caspase-2 is localized to the Golgi apparatus and nuclear membrane in primary CLL cells. Control and brefeldin A-treated (100 ng/mL, 24h) B-CLL cells were cytocentrifuged onto glass slides, fixed, and stained with a monoclonal human anti-caspase-2 antibody and markers for the nucleus (To-Pro-3) and Golgi apparatus (BODIPY-TR-Ceramide). Subcellular localization of caspase-2 was visualized by confocal microscopy using an LSM 510 confocal microscope with a Plan-Neofluar dry 40 ×/0.75 objective lens and a built-in camera (Carl Zeiss, Thornwood, NY). (B) BFA treatment leads to activation of caspases-2, -8, -9, and -3. Lysates were prepared from cells with or without 100 ng/mL BFA treatment for 24 hours. Western blotting was used to evaluate the indicated caspases in primary CLL cells and U266 cells. The antibody used for caspase-3 recognizes the cleaved (active) form only. Actin was used as a loading control. NR = nonrefractory; R = fludarabine refractory.$

Activation of multiple caspases by brefeldin A. (A) Caspase-2 is localized to the Golgi apparatus and nuclear membrane in primary CLL cells. Control and brefeldin A-treated (100 ng/mL, 24h) B-CLL cells were cytocentrifuged onto glass slides, fixed, and stained with a monoclonal human anti-caspase-2 antibody and markers for the nucleus (To-Pro-3) and Golgi apparatus (BODIPY-TR-Ceramide). Subcellular localization of caspase-2 was visualized by confocal microscopy using an LSM 510 confocal microscope with a Plan-Neofluar dry 40 ×/0.75 objective lens and a built-in camera (Carl Zeiss, Thornwood, NY). (B) BFA treatment leads to activation of caspases-2, -8, -9, and -3. Lysates were prepared from cells with or without 100 ng/mL BFA treatment for 24 hours. Western blotting was used to evaluate the indicated caspases in primary CLL cells and U266 cells. The antibody used for caspase-3 recognizes the cleaved (active) form only. Actin was used as a loading control. NR = nonrefractory; R = fludarabine refractory.

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