the induction of apoptosis, 
we analysed the cleavage of important Caspases like Caspase 8, Caspase 7, Caspase 9 and also PARP polymerase) in MCF-7, T47D and MCF10A. As shown in FAE Induced Caspase Activation in Live Cell Model of Caspase Activation Above results indicated that FAE induced apoptosis primarily through mitochondria mediated intrinsic pathway involving Bax mediated mitochondrial permeabilization followed by Caspase 9 and Caspase 3/7 activation. Further, to substantiate the role of Caspases in FAE induced cell death, we have used a FRET based live cell model as described earlier to order XL-518 monitor activated Caspases in live cells. In brief, the breast cancer cell line expressing the FRET probe ECFP-DEVD-EYFP was exposed to FAE at 100 mg/ml and imaged in ratio mode. Upon Caspase activation, the FRET from the donor fluorophore to acceptor was lost with increase in ECFP and decrease in EYFP fluorescence. As shown in FAE – induced Cell Death Involved Generation of ROS Bax Mediated Apoptotic Effect of FAE 7 Bax Mediated Apoptotic Effect of FAE indicates the translocation of Bax to mitochondria, as indicated by the arrows. The representative images collected at indicated time points were used for analysing the percentage positive cells with Bax EGFP onto mitochondria compared to total in the field. Graph showing the percentage of cells undergoing Bax translocation into mitochondria upon FAE treatment. The MCF-Bax-DS Red cells were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22202440 treated as indicated above. BaxEGFP accumulation in mitochondria is indicated in high magnification images with arrows. A magnified merged image of the treated cells is also shown. MCF-7 cells were transfected with Control siRNA or Bax siRNA. After 48 h of transfection, whole cell extract was prepared and immunoblotted for Bax and Actin. The same cells were also stained with Hoechst 33342 after 48 h of FAE treatment to visualize chromatin condensation. The graph is the quantitative representation of the percentage of cells with condensed chromatin in scrambled-transfected and Bax-transfected cells after FAE treatment. doi:10.1371/journal.pone.0040055.g004 Discussion Natural product extracts have been widely tested in the pharmaceutical industry and have been considered as a valuable source of new drugs. As a means of identifying anti-cancer agents, the reverse pharmacology, or the `bedside’ to `bench’ approach has been explored that involves studying medicinal plants that have been traditionally used to treat various ailments. Various studies indicates that Ficus species are widely used in the management of various types of diseases like respiratory disorders, sexual disorders, central nervous system disorders, cardiovascular disorders, gastric problems, skin infections and diabetes etc. Most of the pharmacological studies were aimed on validating its traditional uses. Although modern drug design emphasizes the development of single agents with specific targets, the fact that whole extract has been shown to be more efficacious than its individual components suggests the limitations of this approach. Killing of tumor cells through the induction of apoptosis is now recognised as a strategy for identifying anti-cancer drugs. In the present report, we sought to determine the mechanisms by which the acetone fraction of F.religiosa leaf extract exerts its antiproliferative effect in multiple breast cancer cells. Anti-proliferative effect can be attributed to altered biochemical mechanisms including suppression of cel
