TC-A signaling in native B-CLL cells. (A) After purification of B-CLL cells of patients using immunomagnetic separation, cells were left untreated or treated with 1 or 2.5 μM for 24 hours. Cell lysates were prepared, subjected to immunoblotting, and analyzed for Bax, Bcl-2, caspase-3 and -8, and PARP. As internal loading control for equal amounts of protein, α-tubulin was detected. (B) B-CLL cells of patients were left untreated or pretreated with either the pancaspase inhibitor zVAD-fmk (50 μM) or an inhibitor of caspase-8—like caspases zLETD-fmk (50 μM) for 1 hour followed by the application of TC-A at the indicated concentrations, and incubations continued for 24 hours. Cell lysates were prepared, and caspase-8 was detected by immunoblotting. α-Tubulin was used as internal loading control for equal amounts of protein. In parallel, apoptosis was detected using the annexin V—FITC binding assay. The corresponding percentages of apoptotic cells are presented in the top box of the panel.