Monoclonal anti e cadherin

Monoclonal anti-HIF-1α antibody (RRID:AB_2835328) was purchased from Affinity (USA), monoclonal anti-E-cadherin (RRID:AB_731493) and anti-β-actin antibodies (RRID:AB_306371) were obtained from Abcam (UK), and monoclonal anti-vimentin antibody (RRID:AB_10695459) was purchased from Cell Signaling Technology (CST, USA). Secondary anti-rabbit immunoglobin G (IgG) antibodies were obtained from Santa Cruz (USA). Neofect™ DNA transfection reagent was purchased from Neofect Biotechnologies (China). T-25-cm² flasks for cell culture were purchased from Bever (USA) and Transwell inserts for 24-well plates (6.5 mm diameters and 8.0 µm diameter filters) were purchased from Corning (USA). Sealed hypoxia incubator chambers were obtained from Thermo Fisher Scientific (USA). Other chemicals of analytical grade were obtained from Sigma-Aldrich (USA).

The cells were grown on coverslips in 24-well plates. Before staining, the cells were fixed with 4% formaldehyde for 10 min, followed by 0.2–0.3% Triton X-100 for 10 min and then were blocked in 1% BSA for 1 h. For staining, the cells were incubated with the primary antibodies monoclonal anti-E-cadherin (1:200, Abcam), anti-vimentin (1:200, Abcam) or anti-HIF-1α (1:200, Abcam) for 1 h and then with goat anti-rabbit/mouse secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The nuclei of the cells were counterstained with DAPI (1:10000, Invitrogen). After each step, cells were washed with PBS. The images were acquired by laser scanning microscopy (Zeiss, Jena, Germany).