Western blot Primary antibodies used in Western blot, following manufacturer’s protocols, were anti-MACC1 (Sigma, USA), anti-Met, anti-p-MEK1/2(ser212/ser218), anti-MEK1/2, anti-p-ERK1/2(Thr202/Tyr204), anti-ERK1/2 and anti-MMP2 (Santa Cruz, USA), anti-Akt, anti-p-Akt(Thr308), anti-cyclinD1, anti-cleaved

selleck caspase3 and anti-β-actin (Beyotime Biotechnology, Jiangsu, China). Total protein was extracted using Cell Lysis Buffer for Western and IP (Beyotime Biotechnology, Jiangsu, China), and protein concentration was determined using Bradford assay. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The detection of https://www.selleckchem.com/products/nepicastat-hydrochloride.html hybridized protein was performed by enhanced chemiluminescence kit (Zhongshan Goldenbridge Biotechnology, Peking, China), β-actin was used as a control for normalization. The specific bands were analyzed by Image-Pro Plus 6.0 system.

mTOR kinase assay MTT assay Planted 2 × 104 cells per well into 96-well plates, and added 100 μl medium containing 10% FBS into each well. Five duplicate wells were set up for each group. Cultured cells continuously for 7 days, added 20 μl MTT reagent (5 mg/ml, Sigma, USA) into each well, incubated for another 4 h then aspirated former medium and added 150 μl DMSO. The absorbance of sample was measured by Microplate spectrophotometer (Thermo, USA) at 492 nm. All experiments were done in triplicate. Cell growth curve was plotted versus time by origin 8 software. Monoplast colony formation assay Prepared single cell suspension, seeded about 50, 100, 200 cells of each group into 6-well plates respectively. Added 2 ml medium containing 10% FBS into each well, cultured

cells continuously for one week. Fixated cells with methanol for 5 min, stained cells by hematoxylin for 30 min, counted the numbers of colony (more than 10 cells per colony) under low power lens (× 100) of inverted microscope (OLYMPUS, IX71, Japan), and ADP ribosylation factor calculated the rate of colony formation. Flow cytometry analysis About 1 × 106 cells were treated into single cell suspension with PBS solution, and were prepared following manufacture’s protocol of Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, Jiangsu, China). Then, rates of apoptosis were analyzed with FACScan system (BD, USA). TUNEL assay Dripped single cell suspension onto microscopic slides, incubated cells for 4 h till cells were adherent. Three duplicate slides were set up for each group. Fixated cells by 4% paraformaldehyde for 30 min, blocked cells by 0.3% H2O2 for 30 min, incubated cells with 0.1% Triton X-100 for 2 min, then performed following manufacture’s protocol of In situ cell death detection kit (Roche, German). Selected five visual fields under high power lens (× 400) randomly, counted the numbers of apoptotic body in 100 cells, calculated the rate of apoptosis.