Figure 4.

  Activation of caspases‐9 and 3 and cleavage of PARP induced by apigenin treatment. (a) BC1, BC3 and BCBL cells were treated with and without 25 and 50 μm apigenin for 24 h. Cells were lysed and equal amounts of protein were separated by SDS–PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase‐9, caspase‐3, cleaved caspase‐3 and PARP. (b) Apigenin‐induced activation and cleavage of caspase‐8 and Bid. BC1, BC3 and BCBL1 cells were treated with 25 and 50 μm apigenin as indicated for 24 h. Cells were lysed and equal amounts of protein were separated by SDS–PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against caspase‐8 and Bid. Beta‐actin was used as a loading control. (c) Effect of z‐VADfmk on apigenin‐induced activation of caspase‐3 and PARP. BC1 and BC3 cells were pre‐treated with 80 μm of z‐VAD for 2 h and subsequently treated with 50 μm apigenin for 24 h. Cells were lysed and 20 μg of protein was separated by SDS–PAGE, transferred to PVDF membrane and immunoblotted with antibodies against caspase‐3, cleaved caspase‐3 and PARP. Three independent experiments were performed to confirm the results. Representative blot is shown. (d) Effect of z‐VAD on apigenin‐induced cell death. BC1 and BC3 cells were pre‐treated with 80 μm of z‐VAD for 2 h and subsequently treated with 50 μm apigenin for 24 h. Live and dead cells were scored using trypan blue dye exclusion. The graph displays the mean ± SD of three independent experiments. (e) Apigenin‐induced down‐regulation of cIAP1, XIAP and Survivin expression. BC1, BC3 and BCBL1 cells were treated with and without 25 and 50 μm apigenin for 24 h. Cells were lysed and equal amounts of proteins were separated on SDS–PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against cIAP1, XIAP and Survivin. Beta‐actin was used to measure equal loading. A representative blot of three independent experiments shown.