Fig 1.
Effects of HGF on the phosphorylation of p38 MAPK and MYPT-1 in HuH7 cells.
The cultured cells were stimulated with 30 ng/ml HGF for the indicated periods. The cell extracts were then subjected to SDS-PAGE with subsequent Western blot analysis using antibodies against phospho-specific p38 MAPK, phospho-specific MYPT-1 or GAPDH.
Fig 2.
Effects of SB203580, SP600125, PD98059, deguelin or Y27632 on the HGF-induced migration, and phosphorylation of p38 MAPK (A), JNK (B), ERK (C), AKT (D) and MYPT-1 (E) in HuH7 cells. For the cell migration, the cells were pretreated with the indicated doses of SB203580 (A), SP600125 (B), PD98059 (C), deguelin (D) or Y27632 (E) for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 23 h. The migrated cells were fixed with paraformaldehyde, and stained with DAPI for nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). For the Western blot analysis, the cells were pretreated with the indicated doses of SB203580 (A), SP600125 (B), PD98059 (C), deguelin (D) or Y27632 (E) for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 5 min ((A), (B) and (E)) or 3 min ((C) and (D)). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. * p<0.05 compared to the value of the control cells without HGF. **p<0.05 compared to the value of the cells with HGF alone. N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 3.
Effect of S1P on the HGF-induced migration of HuH7 cells.
The cells were pretreated with the indicated doses of S1P for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 23 h. The migrated cells were fixed with paraformaldehyde, and stained with DAPI for nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar graph). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05 compared to the value of the control cells without HGF. **p<0.05 compared to the value of the cells with HGF alone. Scale bar: 100 μm.
Table 1.
The cell viability of S1P-treated HuH7 cells.
Fig 4.
The HuH7 cell extract was subjected to SDS-PAGE with subsequent Western blot analysis using antibodies against S1PR1, S1PR2, S1PR3, S1PR4, S1PR5 or GAPDH.
Fig 5.
Effect of agonists of S1PR1-5 on the HGF-induced migration of HuH7 cells.
The cells were pretreated with the indicated doses of SEW2871 (A), CYM5520 (B), CYM5541 (C), CYM50260 (D) or A971432 (E) for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 23 h. The migrated cells were fixed with paraformaldehyde, and stained with DAPI for nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar panel). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05 compared to the value of the control cells without HGF. **p<0.05 compared to the value of the cells with HGF alone. N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 6.
Effect of JTE013 on the suppression by S1P of the HGF-induced migration of HuH7 cells.
The cells were pretreated with 1 μM of JTE013 for 10 min prior to 10 μM of S1P treatment for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 23 h. The migrated cells were fixed with paraformaldehyde, and stained with DAPI for nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar panel). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05 compared to the value of the control cells without HGF. **p<0.05 compared to the value of the cells with HGF alone. ***p<0.05 compared to the value of the cells with HGF and S1P pretreatment. N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 7.
Effect of S1PR2-siRNA on the suppression by S1P of HGF-induced migration of HuH7 cells.
The S1PR2-siRNA or negative control siRNA transfected HuH7 cells were pretreated with 10 μM of S1P for 60 min, and then stimulated by 30 ng/ml of HGF or vehicle for 23 h. The migrated cells were fixed with paraformaldehyde, and stained with DAPI for nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel) and counted (bar panel). Each value represents the mean ± SD of triplicate determinations from three independent cell preparations. *p<0.05 compared to the value of the control cells without HGF. **p<0.05 compared to the value of the cells with HGF alone. N.S. designates no significant difference between the indicated pairs. Scale bar: 100 μm.
Fig 8.
A schematic illustration of the regulatory mechanism of S1P in HGF-induced HCC cell migration.
S1P, sphingosine 1-phosphate; HGF, hepatocyte growth factor; HCC, hepatocellular carcinoma, S1PR, sphingosine 1-phosphate receptor.