Ripa protein lysis buffer

Tissues and cells were lysed in RIPA protein lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease inhibitors. The protein concentration was determined using a BCA protein assay (Beyotime Institute of Biotechnology, Jiangsu, China). Next, the proteins were denatured and separated via SDS-PAGE gel (15% for separating LC3B and 6% for separating CD133) and then transferred to nitrocellulose transfer membranes (Whatman, Piscataway, NJ, USA). The membranes were blocked with 5% non-fat powdered milk in phosphate-buffered saline (PBS) for 1 h at room temperature and then incubated with rabbit polyclonal antibody against human LC3B (1:800; cat. no. L7543; Sigma-Aldrich; Merck KGaA), mouse monoclonal antibody against human CD133 (1:500; cat. no. MAB4399-I; EMD Millipore), or rabbit polyclonal antibody against human GAPDH (1:200; cat. no. sc-25778; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The membranes were then washed extensively with TBST and incubated with goat antibody against mouse (1:10,000; cat. no. ab97040; Abcam) and goat antibody against rabbit (1:1,000; cat. no. ab7085; Abcam) for 1 h at room temperature. The signal was detected using an enhanced chemiluminescence system (Thermo Fisher Scientific) in accordance with the manufacturer's protocol. The results were normalized to GAPDH and expressed as relative densities.

DMEM medium (Sigma, St. Louis, MO, USA); pancreatic enzymes for cell dissociation (Sigma); fetal bovine serum (FBS; Sigma); MTS solution (Promega Corp., Madison, WI, USA); rabbit anti-human HIF-2α antibody (Abcam, Cambridge, MA, USA), vascular endothelial growth factor polyclonal antibody, rabbit anti-human extracellular signal-regulated kinase (Erk) antibody and rabbit anti-human phosphorylated Erk (p-Erk) antibody. Mouse anti-human GAPDH antibody, goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP), and goat anti-mouse secondary antibody conjugated to HRP were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); RIPA protein lysis buffer, and BCA protein quantitative kit were from Thermo Fisher Scientific (Waltham, MA, USA). The human osteosarcoma cell line MG-63, was from the cell bank of the Chinese Academy of Sciences (Shanghai, China).

Western blotting was used to measure the expression levels of proteins in this study. SGC cells from each group were lysed with radioimmunoprecipitation assay (RIPA) protein lysis buffer (#89900; Invitrogen; Thermo Fisher Scientific, Inc., USA), and the protein content was determined with a BCA kit (#23252; Pierce; Thermo Fisher Scientific, Inc., USA). Equivalent amounts of protein (40 µg) were separated by SDS-PAGE then transferred to polyvinylidene difluoride (PVDF) membranes and blocked with skimmed milk at room temperature for 1 h. After incubation with primary and secondary antibodies, the protein bands were visualized by enhanced chemiluminescence (#32106; Pierce; Thermo Fisher Scientific, Inc., USA). The following primary antibodies were used at a dilution of 1:1,000: anti-p63 (#39692; 1:1,000; CST, USA), anti-EMA (#14161; 1:1,000; CST, USA), anti-CKpan (#ab7753; 1:500; Abcam, USA), anti-ZEB2 (#97885, 1:1,000; CST, USA), anti-E-cadherin (#3195; 1:1,000; CST, USA), anti-N-cadherin (#13116; 1:1,000; CST, USA), anti-vimentin (#5741; 1:1,000; CST, USA), and anti-GAPDH (#5174; 1:1,000; CST, USA). Grayscale value analysis was performed using ImageJ software (version 1.52a; National Institutes of Health), and GAPDH was used as the loading control.