The control

group was provided by cells incubated with 2 ml of 1640 medium alone. Afterwards cells were collected for further testing. mTOR signaling pathway Western blot 786-O cells and OS-RC-2 cells were lysed in radio-immunoprecipitation assay buffer and equal amounts of the protein extracts (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) for western blotting. The primary antibodies against NOTCH1 (activated Notch intracellular domain), HES-1 (Abcam, Cambridge, MA), and β-actin (Aidlab Biotechnologies Co., Beijing, China) were incubated with

membranes overnight at 4°C. After 3 washes, for 15 min each, in Tris-buffered saline supplemented with 0.1% Tween 20, membranes were incubated with peroxidase-conjugated goat anti-mouse/rabbit IgG antibodies (Aidlab Biotechnologies Co. Beijing, China) for 1 h at room temperature. The bound anti-bodies were visualized by an enhanced chemiluminescence detection system using medical X-ray films. Comparative inhibition of proliferation analysis with CCK-8 assay Cells were seeded in a 96-well plate at approximately 8×104 in a volume of 100 μl/well. Wells were also prepared that contained AZD5153 known numbers of four kinds of cells to be used to create a calibration curve. To measure apoptosis, 10 μl of the CCK-8 solution (Dojindo, Japan) was carefully added to each well of the plate. The plate was incubated for 1–4 h in the incubator during which time the absorbance was measured at 450 nm using a microplate reader at 30, 60, (-)-p-Bromotetramisole Oxalate and 90 min. Transwell assay for cell invasion Cell invasive ability was determined using the Transwell test kit (Corning, NY, USA). Briefly, matrigel was mixed with 1640 medium at a ratio of 1:7 and 100 μl was added to each upper-transwell then placed into the incubator for 1 hour for the mixture to set. Then, 786-O cells were

serum-starved for 12 h in pre-warmed 1640 media alone to eliminate the effects of serum. Twenty-four hours after the application of matrigel, 600 μl of 10% FBS solution was added to the lower transwell. The serum starved cells were resuspended to a density of 2.5×105 in 1640 solution without FBS in a final volume of 1 ml, with or without Marimastat or DAPT. From this, 100 μl was added to each transwell (2.5×104). After 48 h in the incubator, the transwell casters were purged into PBS to remove the non-adherent cells, and then submerged it in 4% paraformaldehyde for 10 min for fixation, and finally replaced in PBS. After the membrane was dried, cells were observed and counted under a microscope (400×).