Cells were incubated at 37 C at 5% CO2. Antibodies directed against phospho Akt, Akt, phospho S6 ribosomal protein, S6 ribosomal protein, phospho MAPK, MAPK, cleaved caspase 3 and actin were from Cell Sig naling. Antibody against CD31 was purchased from BD sellekchem Biosciences. NVP BEZ235 and Inhibitors,Modulators,Libraries sorafenib were purchased from LC Laboratories. Cell count Cells were plated in six well plates Inhibitors,Modulators,Libraries at a density of 100 000 cells well and cultured in DMEM 10% FBS. Twelve hours later, cells were treated with increasing doses of NVP BEZ235, sorafenib, a combination of both or DMSO as a control for 48 or 72 hours. Subsequently, adherent cells were collected and trypan blue negative cells were counted using a Neubauer hemocytometer. MTS proliferation assay Caki 1 or 786 0 cells were plated on 96 well plates at 10000 cells per well and cultured in DMEM 10% FBS.

Twelve hours later, cells were treated with NVP BEZ235 1 uM, sorafenib 10 uM, a combination of both or DMSO as a control. Cellular proliferation was monitored after 48 or 72 hours of treatment with the CellTiter 96 AQueous One Solution colorimetric assay by following the manufacturers instructions. The MTS compound is reduced by Inhibitors,Modulators,Libraries living cells into a formazan product whose quantity is directly proportional to the number of cells in culture. The quantity of formazan product is measured by the amount of 490 nm absorbance. BrdU incorporation assay Cells were plated on coverslips and treated with the indicated inhibitor for 24 hours. 5 bromo 2 deoxyuri dine at a final concentration of 10 uM was added to the culture medium for the last 12 hours.

Sub sequently, cells were fixed with paraformaldheyde for 10 min, washed twice with PBS and incubated with HCl 2 N for 2 min. Cells were extensively washed in PBS and immunocytofluorescence was done with mouse anti BrdU antibody, and the fluorochrome con jugated secondary Inhibitors,Modulators,Libraries antibody against mouse Ig. The nuclei were counterstained with DAPI. Immunostained cells were observed under epifluorescent microscope IX81. BrdU and DAPI positive cells Inhibitors,Modulators,Libraries were counted using a computer assisted image ana lysis station. Results were expressed as the ratio of BrdU to DAPI positive cells. Apoptosis Assay The Cell Death Detection ELISAplus kit was used to measure apoptosis. Caki 1 and 786 0 cells were seeded in 96 well plates at 30,000 cells per well and grown in serum free medium at 37 C.

Twelve hours later, cells were treated with NVP BEZ235, sora fenib, a combination of both, or DMSO as a control, for 24 hours. Subsequently cells were harvested and apoptosis was selleck inhibitor determined following the manufac turers instructions. Results are represented as the mean enrichment factor. Cell cycle analysis Caki 1 and 786 0 cells were treated with NVP BEZ235, sorafenib, a combination of both, or DMSO as a control for 48 hours. Cells were collected and processed for FACS analysis as previously described.