The apoptotic machineries were induced on T315 treatment. (A) CLL cells were incubated in α-IgM coated plates or (C) fibronectin-coated plates or 1 μg/mL VCAM-1 with vehicle or OSU-T315 for 16 hours. Cell lysate was analyzed by immunoblotting and normalized to GAPDH, and the ratio of densitometry to GAPDH was shown beneath the blots. (B) Mcl-1 expression level was quantified according to the results of immunoblotting in the separated conditions of plate-coated α-IgM, 0.5 μg/mL CD40L, 3.2 μM CpG-ODN, or (D) fibronectin-coated plates or 1 μg/mL VCAM-1 along with the treatment of vehicle or 4 μM OSU-T315 for 16 hours. (E) Caspase 3/7 activity was accessed by Caspase-Glo 3/7 Assay Systems (Promega) and normalized to vehicle after 16-hour treatment; 20 μM of Q-VD-OPh served as the negative control, whereas 500 nM flavopiridol served as the positive control. (F) Mec-1 or OSU-CLL cells pretreated with vehicle or 20 μM z-VAD-FMK prior to 4 μM OSU-T315 treatment were analyzed by Annexin V/PI staining after 24 hours.