Protein Expression and Signaling Analysis

Protein extraction and Western blots were conducted and analyzed as previously described [8 (link)] with the antibodies against the following: PTEN, N-Cad, and E-Cad (1:1000; Cell Signaling Technology, MA, USA); Akt and phosphorylated-Akt (phosphorylated on S473), Caspase 3 (1:1000; Abcam, CA, USA); P70S6K and phosphorylated-P70S6K (phosphorylated on S371), mTOR and phosphorylated-mTOR (phosphorylated on S2998; 1:1000; Bioworld, Jiangsu, China); CD63, TSG-101, and Flotillin-1 (1:500; Abcam, CA, USA), and human β-actin (1:5000; Transgene, Beijing, China). The blots were quantified by density relative to β-actin, while the phosphorylation of Akt, mTOR, and P70S6K was determined by density relative to total Akt, mTOR, and P70S6K, respectively. All experiments were performed in triplicate.