Fig. 3

The metastatic potential of GC cells induced by CAFs-derived IL-33 is mediated by the activation of ERK1/2-SP1-ZEB2 pathway via ST2L. a–d Western blot analysis showing the phosphorylation of ERK1/2 in SGC7901 and MKN45 cells treated with medium alone or stimulated as follows: exogenous IL-33 (300 ng/ml), U0126 (20 μM), CAFs supernatants (CAFsu) supplemented with IgG isotype control antibody (3 μg/ml), DMSO; co-cultured with CAFs transfected with IL-33/siRNA or NC/siRNA; CAFsu supplemented with IL-33 neutralizing antibody (3 μg/ml). e Western blot analysis of pERK1/2 in SGC7901 and MKN45 cells incubated with medium alone; exogenous IL-33 (300 ng/ml); U0126 (20 μM); co-culture with CAFs; IL-33 neutralizing antibody (3 μg/ml); and/or ST2L neutralizing antibody (3 μg/ml). f, g The migration and invasion ability of SGC7901 and MKN45 cells after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) and/or U0126 (20 μM); co-culture with CAFs and/or U0126 (20 μM). Histograms show the average cell number per field. (100×; scale bar = 100 μm). h, i QRT-PCR analysis of SP1 mRNA expression in SGC7901 and MKN45 cells incubated in medium alone or stimulation with exogenous IL-33 (300 ng/ml) or CAFs in the presence of IgG isotype control antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). j, k Western blot analysis of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells incubated in medium alone or stimulated by co-culture with CAFs in the presence of exogenous IL-33 (300 ng/ml) or U0126 (20 μM). l Western blot analysis showing the protein expression of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells cultured in medium alone (Mock); or transfected with control plasmid (nc) or SP1-expressing plasmid. m Dual luciferase reporters containing four different lengths of the ZEB2 promoter were co-transfected with SP1-expressing plasmid into 293T cells. Firefly luciferase activity was assessed relative to Renilla luciferase activity. Data are representative of three independent experiments. Densitometry shows relative protein expression. *P <0.05, **P <0.01, ***P <0.001