Fig. 7

The expression of IL6R and IL6 is regulated by NFAT1 in glioma cells. a Expression data of IL6R, IL6 and NFAT1 mRNA in gliomas were downloaded from TCGA dataset and analysed. The expression of IL6R and IL6 were significantly correlated with that of NFAT1. b The expression of NFAT1 in different glioma cells was examined using western blotting. c Effect of NFAT1 knockdown on the expression of IL6R and IL6 was examined by western blotting in M1 and U87 cells. d Effect of NFAT1 overexpression on the expression of IL6R and IL6 was examined by western blotting in T98G and N2 cells. e Double-labelled immunofluorescence staining showing that the downregulation of NFAT1 is accompanied decreased IL6R expression in M1 cells, while upregulation of NFAT1 is accompanied increased IL6R expression in N2 cells. Scale bar =10 μm. f The levels of IL-6 secretion were examined by ELISA in cell-free supernatants from glioma cells after NFAT1 silencing or overexpression. g-h Effect of NAFT1 on IL6R (g) and IL6 (h) promoter activities. Silencing NFAT1 in both M1 and U87 cells significantly reduced the luciferase activity driven by the wildtype IL6R and IL6 promoters compared with control-shRNA. Mutating NFAT1 binding sites resulted in reduced promoter activity compared with the wildtype promoter. NFAT1 overexpression in T98G and N2 cells increased luciferase promoter activities. i Binding of NFAT1 to IL6R and IL6 promoters. Binding of NFAT1 was decreased when NFAT1 was silenced in M1 and U87 cells, while binding was increased when NFAT1 was overexpressed in T98G and N2 cells. j In M1 cells, NFAT1 knockdown significantly inhibited subcutaneous tumour growth. *P < 0.05 and **P < 0.01