Fig 2: Combination treatment with sub-optimal doses of embelin and LY294002 synergistically induces apoptosis in BC cells. (a) AKT siRNA down-regulates expression of XIAP in BC cells. MDA-MB-231 cells were transfected with siRNA targeted against AKT and proteins were isolated and probed with antibodies against p-AKT, AKT, XIAP and GAPDH. (b and c) Combination of embelin and LY294002 at sub-optimal doses synergistically inhibits cell viability and induces apoptosis in BC cells. EVSAT and MDA-MB-231 cells were treated with either alone or in combination of embelin (10 µM) and LY294002 (10 µM) for 24 h. Following 24 h treatment, cells were analysed for cell viability by MTT assays (b) and apoptosis after staining the cells with annexin V/PI by flow cytometry (c). (d) Combination treatment causes inactivation of AKT, down-regulation of XIAP and caspase-dependent apoptosis in BC cells. EVSAT and MDA-MB-231 cells were treated with combination of sub-toxic doses of embelin and LY294002 for 24 h. Following incubation, proteins were isolated and probed with antibodies against XIAP, p-AKT, AKT, Bcl-Xl, caspase-9, caspase-3, PARP and GAPDH

Fig 3: SAHA affects the expression of suvivin and XIAP at the transcriptional level. (A) MCF7 and (B) MDA-MB-231 cells were treated with various concentrations of SAHA for 24 h. The relative amount of survivin and XIAP mRNA transcripts present in cells was analyzed by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with SAHA vs. without SAHA (negative control) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 cells were treated with SAHA for 24 h and expression of p53 was determined by Western blotting. (D) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 48 h. Expression of different proteins was analyzed by Western blotting. Signals in the survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDACs on the expression of survivin. (E) MCF7 cells were transfected with scramble, HDAC2, 3, or 6 siRNA for either 24 or 48 h. Relative amount of survivin mRNA transcripts present in cells was determined by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with HDAC2, 3, or 6 siRNA vs. scramble siRNA is denoted by either “*” (p < 0.05) or “**” (p < 0.01).

Fig 4: Embelin/LY294002 combination inhibits growth of MDA-MB-231 Xenografts. Female nude mice at 6 weeks of age were injected S.C. with 10 million MDA-MB-231 cells. After one week, mice were divided into four groups; the first group only received DMSO vehicle alone; the second and third group were treated with either embelin (10 mg/kg) or LY294002 (10 mg/kg) and the fourth group were treated with combination of embelin and LY294002. (a) Volume of each tumour was measured every week. The average (n = 4) tumour volume of mice was calculated, * p < 0.05 inhibition of MDA-MB-231 tumour growth by combination of embelin and LY294002. (b) After 4 weeks of treatment, mice were sacrificed and tumour weights were measured,*p < 0.05 compared to vehicle-treated mice by Student’s t-test. (c) Representative tumour images of vehicle, embelin, LY294002 and combination of embelin and LY294002 treated mice. (d) Whole cell lysate from mice treated with different inhibitors were isolated and 10 µg protein were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against XIAP, p-AKT, AKT, Bcl-Xl, Bcl-2, caspase-3 and Beta-actin

Fig 5: (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 GAPDH