N cad

Cells and human RCC tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China), supplemented with protease inhibitors (Roche, Shanghai, China) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Roche), at 4°C for 30min. The cell supernatants were extracted after centrifugation for 15 min at 14,000 rpm, then the concentration of the protein was determined using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). Proteins from tissues and cells were separated using 10% SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, USA), blocked for 2h with 5% nonfat milk at room temperature, and incubated with primary antibodies at 4°C overnight. After that, the membranes were washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, CA, USA), and antibodies against β-actin served as a negative control. Rabbit monoclonal antibodies (1:1000) against TGF-β1, E-cad, N-cad, Vimentin and MMP-9 (Cell Signaling Technology, USA) were used in Western blot analysis according to the manufacturer’s instructions.

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.

The differentiated THP1 cells were lysed with 1 X RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and stored in aliquots at -20°C until use. Twenty micrograms of cell lysates were denatured by boiling, and separated by SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane (BioRad). The membranes were blocked using 5% non-fat dry milk for 1 h at room temperature and probed with antibodies overnight. After incubated with IgG horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Beverly, MA) for 2 h at room temperature, the immunoblots were developed using the enhanced chemiluminescence (ECL) reagent (Cell Signaling, Beverly, MA) and visualized using a FluroChemQ processor (Proteinsimple, Santa Clara, CA). The antibodies used in this study include CD206, ERK, pERK. AKT, pAKT, foxo1, Glut1, N-cad and skp2, which were obtained from Cell Signaling Technologies.

IHC staining was performed using a Leica BOND III automated system. Anti‐FAP antibody (ab207178, 1:400) was used for the IHC staining of tumor tissues from patients with glioma. Immunostained slides of TMA sections were scanned using a Leica Aperio AT2 scanner (at 400× magnification) and analyzed using a Leica Aperio ImageScope v12.3. The automated algorithm scored the staining of each tissue core as negative (0), weak (1+), moderate (2+), or strong (3+) according to the scoring criteria threshold. The algorithms also determined the percentage of positive staining. Then, the H‐score of each case was established using the formula H‐score = 1 × (percentage of 1+ cores) + 2 × (percentage of 2+ cores) + 3 × (percentage of 3+ cores). Thus, the H‐score ranged from 0 to 300.¹³ FAP (ab207178), Ki‐67 (ab15580), E‐cad (Cell Signaling Technology, #14472), and N‐cad (Cell Signaling Technology, #13116) antibodies were used for the IHC staining of subcutaneous tumors from BALB/c athymic nude mice.

The protocol used for western blotting is described elsewhere.^{22 (link)} Total cellular protein was extracted in lysis buffer by radioimmunoprecipitation assay. The protein concentration was determined by using the bicinchoninic acid kit (Biyuntian, Jiangsu, China). Load the same amount of protein onto 10% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis, the protein was blotted onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% skim milk and incubated with the primary antibody overnight at 4 °C, and then treated with secondary antibody at 37 °C for 2 h. The primary rabbit anti-human antibodies used were as follows: ECAD (Cell Signaling Technology), NCAD (Cell Signaling Technology), vimentin (Proteintech Group), MMP1 (Cell Signaling Technology), MMP3 (Abcam), IGFBP3 (Abcam), β-catenin (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-Akt (Cell Signaling Technology), GSK-3β (Cell Signaling Technology), phospho-GSK-3β (Cell Signaling Technology), TBP (Proteintech Group) and β-actin (Cell Signaling Technology).