Fig. 3

Embelin causes activation of mitochondrial apoptotic pathway via inactivation of AKT in BC cells. (a) Embelin treatment causes inactivation of AKT, Bad and down-regulation of Bcl-2 and Bcl-Xl in BC cells. EVSAT and MDA-MB-231 cells were treated with 25 and 50 μM embelin for 24 h and proteins were isolated, separated on SDS-Page and probed with antibodies against p-AKT, AKT, p-Bad, Bcl-2, Bcl-Xl and GAPDH. (b) Embelin induced inactivation of AKT and down-stream targets are confirmed by siRNA transfection against XIAP. EVSAT and MDA-MB-231 cells were transfected with either non-specific scrambled siRNA or specific siRNA targeted against XIAP for 48 h. Following transfection, proteins were isolated and probed with antibodies against p-AKT, AKT, p-Bad, Bcl-2, Bcl-Xl and GAPDH. (c) Change in mitochondrial membrane potential determined by JC1 staining following Embelin treatment in BC cells. BC cells were treated with 50 μM of embelin for 24 h and following treatment, cells were stained with JC1 and analysed for red stained cells (intact mitochondria) and green stained cells (damaged mitochondria) by flow cytometry. (d) Embelin-induced release of cytochrome c in BC cells. MDA-MB-231 cells were treated with 25 and 50 μM Embelin for 24 h. Following treatment, mitochondrial free cytosolic extracts as well as mitochondrial extracts were isolated and probed with antibodies against cytochrome c, Cox IV and GAPDH. (e) Embelin treatment also down-regulates expression of IAPs in BC cells. EVSAT and MDA-MB-231 cells were treated with embelin for 24 h and proteins were probed with antibodies against cIAP1 and Survivin. GAPDH was used as a loading control