Fig. 4

Activation of caspase-9, caspase-3, and cleavage of PARP induced by bortezomib treatment in CML cells. a Bortezomib mediated activation of caspase cascade in CML cells. AR230 and K562 cells were treated with and without 10, 25 and 50 nm of bortezomib for 24 h. Cells were lysed and 25 μg of proteins were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, cleaved caspase-3, PARP and GAPDH. b Bortezomib-induced activation of caspase-3 and PARP cleavage in K562 cells was determined by flow cytometry as described in “Methods” section. *p < 0.05, ***p < 0.001. c Effect of z-VAD-fmk on bortezomib-induced apoptosis and activation of caspase-3. K562 cells were pre-treated with 80 μM z-VAD/fmk for 2 h and subsequently treated with 25 nm for 24 h. Cells were divided in two parts. In one part apoptotic cells were measured with fluorescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry (upper panel). The other part of the cells were lysed and 20 μg protein were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, pro-caspase-3, cleaved caspase-3, and GAPDH (lower panel). d Bortezomib down-regulates expression of XIAP in CML cells. K562 and AR230 cells were treated with 10, 25 and 50 nm bortezomib for 24 h. Following incubation, cells were harvested and proteins were isolated separated on SDS-PAGE and immunoblotted with antibodies against XIAP, cIAP1, Survivin and GAPDH as indicated (representative of several experiments)