Eighteen microliters of master blend containing cDNA and SYBR Green was included with 2uL of the 100uM forward and reverse primer. PCR and detection was performed in an ABI prism 7000 thermocyler. Results were quantitated using the CT method. MAPK activation Primer sequences are given or have already been described previously. 105 cells were fixed from the dropwise addition of 4. 5mL of ice cold 95-pound ethanol all through slow vortexing and placed at 4 C for 24 hours. Washed cells were resuspended in 300uL of PBS 2% FBS containing 10ug/mL of propidium iodide and 250ug/ml RNAase A for thirty minutes just before analysis. 5,000 single cell activities were captured using a flow cytometer and analyzed using Modfit software. Mammalian target of rapamycin signaling plays a key role in protein translation, cell growth, autophagy and k-calorie burning. Activation of phosphatidylinositol 3 kinase /Akt/mTOR signaling plays a role in the pathogenesis of many tumor types. Rapamycin is an allosteric inhibitor of mTOR. Rapamycin analogs, have been FDA-APPROVED for treating neuroendocrine tumors, renal cell carcinoma and subependymal giant cell astrocytoma related to tuberous sclerosis, and Cellular differentiation have very encouraging clinical benefit in other tumor types including breast and endometrial cancer. However, rapalogs demonstrate objective responses in just a subset of patients and unfortuitously responses are frequently short lived. Therefore, there is a pressing need to spot predictors and pharmacodynamic markers of rapamycin reaction, and mechanisms of treatment resistance. Activation of Akt is proposed to become a predictor of rapamycin answer. Rapamycin and its analogs have been shown to cause Akt activation. Insulin like growth factor I and insulin dependent induction of the PI3K/Akt process Ganetespib dissolve solubility contributes to feedback inhibition of signaling due to mTOR/S6K mediated phosphorylation and degradation of IRS 1. Rapamycin induced Akt activation is primarily related to the increased loss of this negative feedback loop. This feedback loop activation of Akt was not only noticed in vitro, but was also seen in a Phase I clinical trial of rapamycin analog everolimus. There’s concern that Akt service may restrict the antitumor efficacy of rapamycin and analogs. The reason for this study was to find out whether PI3K pathway mutations or Akt activation at baseline is a predictor of rapamycin sensitivity, and whether rapamycin induced Akt activation is related to resistance to rapamycin and analogs in vitro and in the clinic. Cell lines used are described in the Supplementary Techniques. Cells were plated in triplicate at densities of 500 to 5,000 cells per well based on growth faculties of the cell lines. After sticking immediately, rapamycin response was established by treating with six levels based on a 10 fold dilution series.