Fig 2: Effects of hypoxia induced by CoCl2 on HIF-1α expression, and the upregulation of fascin-1 expression by HIF-1α under hypoxia in FaDu cells. (A) Expression of HIF-1α was observed following treatment with 0, 25, 50, 100, 200 or 400 µmol/l of CoCl2 for 6, 12 or 24 h in FaDu cells. Western blotting revealed that HIF-1α expression was upregulated in a concentration-dependent manner. β-actin was used as an internal reference. (B) Expression of HIF-1α in FaDu cells was observed following treatment with 200 µmol/l CoCl2 for 0, 3, 6, 12 or 24 h. Western blotting revealed that CoCl2 induced HIF-1α expression in a time-dependent manner, and HIF-1α expression peaked at 12 h in FaDu cells. β-actin was used as an internal reference. (C) Viability of FaDu cells treated with 0, 25, 50, 100, 200 or 400 µmol/l CoCl2 for 6, 12 or 24 h, as determined by Cell Counting kit-8 assay. No significant difference was detected in cell viability at concentrations ranging between 0 and 200 µmol/l. However, following treatment with 400 µmol/l CoCl2, cell viability was significantly decreased. **P<0.01, ***P<0.001 vs. 0 µmol/l. Western blotting and semi-quantitative analysis revealed that (D) compared with under normoxia, hypoxia induced by treatment with 200 µmol/l CoCl2 for 12 h upregulated HIF-1α and fascin-1 expression in FaDu cells. Student's t-test was used for analysis. (E) HIF-1α knockdown reduced the expression of fascin-1 under hypoxia post-transfection with HIF-1α siRNA for 48 h in FaDu cells. One-way ANOVA was used for analysis. β-actin was used as an internal reference. Protein expression was semi-quantified using ImageJ software, and the CoCl2 (-) group was set at 1. Data are presented as the mean ± SD. *P<0.05, **P<0.01. CoCl2, cobalt chloride; HIF-1α, hypoxia inducible factor-1α; siRNA, small interfering RNA.

Fig 3: Fascin-1 promotes the invasion and migration of FaDu cells by upregulating MMP-2 expression. MMP-2 expression was examined by western blot analysis and semi-quantitative analysis revealed that (A) compared with in the control cells, MMP-2 expression was significantly downregulated in FaDu cells transfected with fascin-1 siRNA. (B) Compared in with the control cells, MMP-2 expression was significantly upregulated in FaDu cells transfected with fascin-1 expression plasmids. β-actin was used as an internal reference. MMP-2 expression was semi-quantified using ImageJ software. These experiments were performed in triplicate, and the bar graphs were obtained from three experiments. All values were normalized to cki or GV144 and paired Student's t-tests were used for analysis. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. Cki or GV144. Cki, control-siRNA; MMP-2, matrix metalloproteinase-2; siRNA, small interfering RNA.

Fig 4: Immunochemical analysis of fascin-1 expression in HSCC. Fascin-1 protein expression in (A-D) paracarcinoma and (E-H) HSCC tissues. Higher magnification images of the boxed areas in (A), (C), (E) and (G) are shown in (B), (D), (F) and (H), respectively. (A, B, E and F) Negative expression of fascin-1 in paracarcinoma and HSCC tissues. (C, D, G and H) Positive expression of fascin-1 in paracarcinoma and HSCC tissues. (D) In the paracarcinoma tissues, fascin-1 revealed weak or partly moderate staining restricted to the basal layers. (H) In HSCC tissues, fascin-1 expression at the invasive front of the tumor was markedly stronger than in the other regions. Magnifications, x100 and x200. HSCC, hypopharyngeal squamous cell carcinoma.

Fig 5: Effect of fascin-1 on cell migration and invasion in FaDu cells. Wound-healing assay revealed that (A) fascin-1-silenced FaDu cells migrated more slowly than the control cells; whereas (B) fascin-1-overexpressed FaDu cells migrated more quickly compared with the control cells (magnification, x100). Graphs show the fold change of wound healing margin at 24h, compared with the wound at 0 h. Data are presented as the mean ± SD from three independent experiments. Migration assay demonstrated that (C) the number of migrated cells was obviously decreased in fascin-1-silenced group compared with the control group; whereas (D) the number of migrated cells was significantly increased in the fascin-1-overexpressed group compared with the control group. Invasion assay demonstrated that (E) the number of invasive cells was obviously decreased in the fascin-1-silenced group compared with the control group; whereas (F) the number of invasive cells was significantly elevated in the fascin-1-overexpressed group compared with the control group. Magnification, x100. Data are presented as the mean ± SD. **P<0.01; ***P<0.001 vs. Cki or GV144. Cki, control-siRNA.