Fig. 4

GC cell-derived TNF-α promotes the expression of IL-33 in CAFs. a, b IL-33 mRNA expression by CAFs was assessed by QRT-PCR after culture in medium or activation with the following stimuli: exogenous TNF-α (50 ng/ml); co-culture with SGC7901 or MKN45 cells; IgG isotype antibody (3 μg/ml); TNF-α neutralizing antibody (3 μg/ml). c, d IL-33 protein expression in the supernatants of CAFs was assessed by ELISA after activation as in a and b. e–g IL-33 mRNA expression by CAFs was assessed by QRT-PCR after culture in medium or activation with the following stimuli: exogenous TNF-α (50 ng/ml); supernatants from MKN45 cells (MKN45su) or SGC7901 cells (SGC7901su); isotype antibody (3 μg/ml); TNFR1 neutralizing antibody (10 μg/ml); TNFR2 neutralizing antibody (10 μg/ml). h, i The nuclear translocation of p65 in CAFs was detected by IF after culture in medium alone or activation with the following stimuli: exogenous TNF-α (50 ng/ml); supernatants from SGC7901cells (SGC7901su) or from MKN45 cells (MKN45su); SN50 (5 μM); PDTC (5 μM); IgG isotype antibody (3 μg/ml); TNFR1 (10 μg/ml) or TNFR2 (10 μg/ml) neutralizing antibodies. Histograms displaying the number of p65 nuclear translocation in each group. j, k IRF-1 mRNA expression by CAFs was assessed by QRT-PCR after activation as in a and b. l IRF-1 mRNA expression by CAFs was assessed by QRT-PCR after culture in medium alone or stimulation with PDTC, TNF-α or DMSO. m IL-33 mRNA expression by CAFs was assessed after activation as in l. n The mRNA expression of IL-33 and IRF-1 in CAFs was explored by QRT-PCR after IRF-1 siRNA transfection. o Dual luciferase reporters containing six different truncations of the IL-33 promoter region were co-transfected with IRF-1-expressing plasmid into 293T cells. Firefly luciferase activity was detected relative to Renilla luciferase activity. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001