Fig. 2

CSC promoted EMT, migration and invasion in OCSS cells, and increased tumor-associated tube formation of HUVECs. OSCC cells were treated with/without 120 μg/ml CSC for 48 h. A The expressions of BiP and vascular endothelial growth factor (VEGF) were analyzed by Western blot analysis. B The amount of VEGF in conditioned media (CM) derived from OSCC was examined by ELISA. C Representative images of epithelial–mesenchymal transition (EMT) morphological changes in OSCC cells after the indicated treatments (Scale bar, 100 μm). D The expressions of epithelial markers (E‐cadherin and ZO-1) and mesenchymal markers (fibronectin and vimentin) were detected by Western blot analysis. E The migratory ability of OSCC cells was evaluated using a wound-healing assay. Representative images were taken at 0 and 12 h after wound scratching (Scale bar, 100 μm). Solid black lines indicate wound borders. Quantification of wound closure was determined using ImageJ software. The ability of migration was calculated as the average reduction in area at 12 h compared to 0 h. F The invasion ability was determined using a Transwell invasion assay. OSCC cells were seeded into the upper chamber of Matrigel-coated inserts. After 24 h, the invaded cells were fixed with methanol and stained with propidium iodide (PI). Representative images are shown (Scale bar, 50 μm). Quantification of the invaded cells was performed using ImageJ software. G Tube formation activities of HUVECs were evaluated using a tube formation assay. HUVECs were seeded in 96-well culture plates pre-coated with ECMatrix and incubated with CM harvested from OSCC cells with/without CSC treatment. After 6 h, tube formation was observed under an inverted microscope and photographed. Representative images are shown (Scale bar, 100 μm). Quantitative analysis of total tube length was conducted using an angiogenesis analyzer for ImageJ software. GAPDH was used as the internal control. All data are presented as the mean ± SEM. SEM, error bar. *P < 0.05 by Student’s t test