Fig. 2From: XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis(a) Embelin inhibits cell viability in BC cells. (a) Breast cancer cells; CAL-120, EVSAT, MCF-7 and MDA-MB-231cells were treated with increasing doses of embelin ranging between 0 and 50 μM concentration. Cell viability assays were performed using MTT as described in Materials and methods. The graph displays the mean +/− SD (standard deviation) of three independent experiments with replicates of six wells for all the doses and vehicle control for each experiment * p < 0.01 and ** p < 0.001, statistically significant (Students t-test). (b) Embelin treatment induces apoptosis in BC cells. BC cells were treated with increasing doses of embelin for 24 h and cells were analysed for apoptosis after staining with annexin V/PI dual staining by flow cytometry. (c) Embelin inhibits expression of XIAP and induces cleavage of caspases-9, −3 and PARP in BC cells. EVSAT and MDA-MB-231 cells were treated with 25 and 50 μM embelin for 24 h. After cell lysis, equal amounts of proteins were separated on SDS-PAGE, and immunoblotted with antibodies against XIAP, caspase-9, caspase-3 and GAPDH as indicated. (d) XIAP siRNA transfection down-regulates XIAP expression and activates caspases in BC cells. EVSAT and MDA-MB-231 cells were transfected with either scrambled siRNA or specific siRNA against XIAP for 48 h. Following transfection, proteins were isolated and probed with antibodies against XIAP, caspase-9, caspase-3, PARP and GAPDH. (e) Embelin down-regulates XIAP transcript in BC cells. EVSAT cells were treated with 25 and 50 μM Embelin for 24 h and RNA were isolated, reverse transcribed into cDNA. Serial dilutions of untreated EVSAT cells cDNA were used to generate a standard curve for GAPDH and XIAP expression. Following treatment, quantitative RT-PCR was performed on cDNA of PTC cells treated with and without 25 and 50 μM Embelin for the expression of XIAP and GAPDH. Absolute qRT-PCR analysis was performed using ABI-7900HT Fast Real-Time PCR system. The results were plotted on a bar graph and standard deviation calculated. Three replicates for each sample were used. (f) Embelin-induced apoptosis in BC cells is caspase dependent. MDA-MB-231 cells were either pre-treated with universal caspase inhibitor, zVAD-fmk for 3 h followed by treatment with embelin for 24 h. Proteins were isolated and probed with antibodies against caspase-9, caspase-3, PARP and GAPDHBack to article page