cells 002, rapamycin, NVP BEZ235 and AZD6244, the cells were lysed using standard procedures. DMSO used alone to control cells. The following primary Ren rabbit anti-human Antique rpern were used: PARP phosphorylated Ser473 phosphorylated AKT p70S6K Thr389 1:1000. Mouse anti-human anti-caspase 2 was used at a concentration of 1:1000. A mouse monoclonal Elvitegravir Antique Body against actin was used at 1:10000 normalization protein gel loading. Clonogenic assays cells were sown at a density of 500 cells per well in 6-well plates t and adhere overnight. The cells were treated with NVP BEZ235 incubated at concentrations of 1, 10 and 40 M ? for seven days. Association studies were performed with NVP BEZ235 at 5, 20, and 50 M and ? AZD6244 50, 500, and 5000 Mr. ? Middle and drugs were replaced on the eighth day.
On day 11, the cell colonies were fixed with glutaraldehyde and 1 to 25 methanol, Customized rbt With 0.5 crystal violet and colonies counted consisting of more than 50 cells Hlt. Experiments were performed in triplicate and the results averaged. Cytotoxicity Was t relative to Kontrollv Lkern determined. Expression of mTOR WZ8040 results in human melanoma tumors and expression of PI3K Co To assess the expression profiles of mTOR in melanoma samples, found Rbt 230 prim we Ren and 293 metastatic melanoma with an antique Body against mTOR. The intratumoral heterogeneity t in the expression of mTOR, two different melanoma TMA, each were meant containing a core of a different area of the tumor, each patient stained. 1A and 1B respectively show examples of strong and weak immunoreactivity t of mTOR, which in the cytoplasmic region, the nuclear compartment was not expressed.
Expression in the two tables were closely correlated, and scores were averaged for each case, to generate uses a composite score for the analyzes. AQUA scores were 11.12 to 69.36. mTOR expression was not survive in patients with primary acids or metastatic connected. We investigated the relationship between the expression of mTOR and expression already ver Ffentlicht and p85 subunits of PI3K p110. With the method of Spearman, s correlation coefficient nonparametric rank, a strong association was found between the expression of p110 and mTOR expression, w Was during a weak association between the levels of mTOR and p85 found.
The synergy between PI3K and mTOR inhibition using concentrations of 5, 25 and 50M of the PI3K inhibitor LY294002, we examined the synergy with various concentrations of rapamycin in five melanoma cell lines. Synergy was observed in all five cell lines at a concentration of 5 M LY294002 with three concentrations of rapamycin. No synergy was at h Heren concentrations of LY294002 observed. Interestingly, the decrease in Lebensf Seen capability with the addition of all concentrations of rapamycin Similar Lebensf Capability with the addition of 1 M rapamycin ? LY294002 was Similar 1M seen. In mutant and wild-type BB Raf Raf-cell line, we evaluated the effects of rapamycin and LY294002, alone or in combination, on the