Fig. 3

BiP modulated CSC-induced invasion of OSCC cells and tumor-associated angiogenesis by upregulating VEGF. YD38 and SCC25 cells transfected with either non-targeting siRNA or BiP siRNA were treated with 120 μg/ml CSC for 48 h. A and B The mRNA and protein expressions of BiP and VEGF were detected by qRT-PCR and Western blot analysis, respectively. C ELISA was used to detect the amount of VEGF in conditioned media (CM) harvested from OSCC cells. D The ability of cell invasion was examined using a Transwell invasion assay. OSCC cells after the indicated treatments were seeded into the inserts pre-coated with Matrigel and incubated for 24 h. Subsequently, the cells were fixed with methanol and stained with propidium iodide (PI). Representative images are shown (Scale bar, 50 μm). The PI-stained cells were quantified using ImageJ software. E The effect of OSCC cell-derived CM on the angiogenicity of HUVECs was examined using a tube formation assay. HUVECs in CM from OSCC cells were seeded in 96-well culture plates containing ECMatrix. Tube formation of HUVECs was observed and imaged after 6 h (Scale bar, 100 μm). Total tube length was quantified using an angiogenesis analyzer for ImageJ software. F The downstream role of VEGF in BiP-mediated tumor-associated tube formation of HUVECs was evaluated by addition of 10 ng/ml of VEGF recombinant protein into CM harvested from BiP-silenced cells after treated with CSC. HUVECs incubated with OSCC-derived CM with/without VEGF addition were seeded in 96-well culture plates containing ECMatrix. After 6 h, tube formation of HUVECs was observed and photographed under a microscopy equipped with a camera (Scale bar, 100 μm). Total tube length was determined using an angiogenesis analyzer for ImageJ software. SC, non-targeting siRNA-transfected cells. BiP-KD, BiP siRNA-transfected cells. GAPDH was used as the internal control. V, VEGF recombinant protein. All data are presented as the mean ± SEM. SEM, error bar. *P < 0.05 by one-way ANOVA followed by Bonferroni’s post hoc test