Parental MCF 7 and MCF7/MR cells were seeded in 96 well plates and developed for 3 days to allow for the synthesis of EVs. Cells were then exposed for a 90 min pre incubation with GW0742 or 1 h pre incubation with Ko143, accompanied by co incubation with increasing levels of MR or topotecan for additional 5 h or 72 h, respectively. In the event of MR cytotoxicity, viable cell numbers were determined after 72 h of treatment with MR. To determine the cytotoxic effect of LY294002 on MCF 7 and MCF 7/MR cells, cells were subjected to various levels of LY294002 for 6. 5 h following 3 washes with fresh medium and incubated for additional 72 h ahead of cell growth research. Drug concentrations necessary to inhibit cell growth by 50% were identified and compared between the cell lines. Western blot analysis was preformed with rat anti ABCG2 antibody as described previously, to examine the cellular expression degrees of ABCG2 following LY294002 or Ko143 therapy. Similarly, an purified rabbit polyclonal antiserum for the a of Na /K ATPase and anti actin antibody were used as a sign of packing differences. We postulated that the PI3K Akt signaling pathway might control the differential sorting of ABCG2 towards the membrane of EVs in MCF 7/MR cells. Being a first step towards this end, we examined whether LY294002, Ribonucleic acid (RNA) a recognised Akt effector protein chemical, could block the activation of the PI3K Akt signaling pathway via inhibition of its phosphorylation. Ergo, EVs building MCF 7/ MR cells were stimulated with EGF for different times in the presence or absence of LY294002, following which phosphorylatedAKT protein levels were based on Western blot analysis using a pAKT specific antibody. After 5 min of stimulation with EGF, pAKT protein levels were already 5fold improved as compared to non stimulated cells. On the other hand, when cells PF 573228 were pretreated for 90 min with LY294002 just before EGF stimulation, AKT phosphorylation was substantially plugged. We determined the extent of inhibition of AKT phosphorylation by dividing the values of pAKT levels following LY294002 treatment by the values obtained with untreated controls, after 5 min of LY294002 treatment, continuing pAKT levels were 23%, while by the end of 30 min, only 15-20 of initial pAKT levels were detected. Hence, LY294002 achieved a inhibition of AKT phosphorylation. Notably, the 20 mM concentration of LY294002 was plumped for according to multiple studies described in the literature which used this Akt signaling pathway inhibitor in various cell types including in vivo isolated mouse hematopoietic stem cells together with SP of glioma stem cells and renal epithelial LLC PK 1 cells.