Fig 2: The effects of rhIL-33 on CCA cell migration. (A) The endogenous or basal expressions of IL-33, ST2L and sST2 in cell lysate and conditioned-medium (CM) of CCA cell lines and CAFs using Western blot analysis. (B and C) The densitometry analysis of protein bands in the Western blot analysis. Bar graphs represent mean ± SD of band intensity normalized with the band intensity of ß-actin as the internal control. (D) Wound healing migration assay of parental KKU-055 compared to IL-33 KD cells at 18 h and 24 h. (E-G) The rhIL-33 effect on (E) KKU-055 (F) KKU-100 and (G) KKU-213 migration using the wound healing assay. Graphs represent mean ± SD. *P-value <0.05 and **<0.01 represent the comparisons to those of normal control without rhIL-33 treatments.

Fig 3: Pulmonary hypertension parameters in SUHX mice. Developed right ventricular pressures in (a) C57Bl/6J, (b) ST2-/-, and (c) MYD88-/- mice in response to SUHX and DMSO/RA. Right ventricle contractility [(RV) + dP/dt] in response to SUHX and DMSO/RA treatments in (d) C57Bl/6J, (e) ST2-/-, and (f) MYD88-/- mice. Right ventricle relaxation [(RV) - dP/dt] in response to SUHX and DMSO/RA in (g) C57Bl/6J, (h) ST2-/-, and (i) MYD88-/- mice. Values are presented as mean ± SD (n = 15 male, n = 8 female DMSO/RA; n = 14 male, n = 6 female SUHX). *Significant difference between SUHX and DMSO/RA within a sex (p < 0.05)

Fig 4: Model for the epithelial–stromal interactions in the tumor microenvironment of GC. a IL-33 released from CAFs promotes the migration, invasion and EMT of GC cells via the ST2L-ERK1/2-SP1-ZEB2 pathway. b GC cell-derived TNF-a upregulates IL-33 expression in CAFs via the TNFR2-NF-?B-IRF-1 pathway

Fig 5: 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