Lumiliximab induces activation of Bax in CLL cells. (A) CLL cells treated with isotype control or lumiliximab for 4 hours were fixed, permeabilized, stained with anti-Bax NT polyclonal antibody followed by secondary anti–rabbit Ig-FITC, and analyzed by confocal microscopy. (B) Increasing levels of active Bax were detected by intracellular flow cytometry at 2 and 4 hours after treatment with lumiliximab but not in cells treated with isotype control. (C) Lumiliximab treatment induces the translocation of cytochrome c. Cytosol and mitochondria-enriched membrane fractions were prepared as described in “Subcellular fractionation” from isotype control or lumiliximab-treated cells. Samples were analyzed by immunoblot with antibodies against cytochrome c. (D) Summary of caspase inhibition data showing mean plus or minus SD (n = 3). (E) Caspase-9 activation induced by lumiliximab was blocked in the presence of caspase inhibitors z-VAD-fmk (ZVAD) and z-LEHD-fmk (LEHD), confirming the activity of the caspase inhibitors used in these experiments.