Fig. 6: IGF2BP3 regulates IRF1 via its upstream transcription factor NFAT1.

A The protein expressions of IRF1, p-STAT1 and STAT1 in MKN-45 cells after treated with indicated dose of IFN-γ for 48 h were examined by western blot analysis. B The protein expressions of IRF1 and p-STAT1 in shNC and shIGF2BP3 MKN-45 cells were examined by western blot analysis. C The protein expressions of IRF1, p-STAT1 and STAT1 in shNC and shIGF2BP3 MKN-45 cells after treated with FAMP for 24 h were examined by western blot analysis. D Overlapping genes of TFs of IRF1 predicted by the PROMO (Maximun matrix dissimilarity rate: 10%), downregulation (|fold change| ≥ 1.5) in shIGF2BP3 MKN-45 cells compared with shNC MKN-45 cells based on Ribo-seq data and upregulation in GC tissues based on the GEPIA were identified; E The protein expressions of SP1 and NFAT1 in shNC and shIGF2BP3 MKN-45 and AGS cells were examined by western blot analysis, respectively. F The expressions of IRF1 in shNC and shIGF2BP3 MKN-45 cells after transfected over expression plasmid targeting NFAT1 or vector control for 48 h were measured by western blot analysis, respectively. G shNC and shIGF2BP3 MKN-45 cells were co-transfected with NFAT1 over expression or vector control plasmid, pGL3-Basic-IRF1 promoter reporter plasmid and pRL-TK plasmid for 24 h. The promoter activities were determined as a relative signal of F-luc divided by R-luc. H Protein expressions of NFAT1 in MKN-45 cells were measured by western blot analysis after transfected siRNA or over expression plasmid targeting NFAT1 for 48 h, respectively. I After transfected siRNA or over expression plasmid targeting NFAT1 for 48 h, the relative mRNA expressions of IRF1 in MKN-45 cells were investigated by qRT-PCR analysis. J After transfected siRNA or over expression plasmid targeting NFAT1 for 24 h, MKN-45 cells were co-transfected with pGL3-Basic-IRF1 promoter reporter plasmid and pRL-TK plasmid for 24 h. The promoter activities were determined as a relative signal of F-luc divided by R-luc. K The consensus sequences of TF binding sites in IRF1 promoter region were predicted by motif analysis from the JASPAR database. L The specific TF binding sites in IRF1 promoter region were predicted. M The binding between NFAT1 protein and specific TF binding sites in IRF1 promoter region in MKN-45 cells were measured by ChIP-qPCR assay. N Schematic representation of mutated (TCC to AGG) promoter region of pGL3-Basic-IRF1 promoter reporter plasmid to investigate the role of specific TF binding sites on IRF1 promoter activities. O shNC and shIGF2BP3 MKN-45 cells were co-transfected with wild-type pGL3-Basic-IRF1 promoter reporter plasmid or mutated (TCC to AGG) promoter plasmid and pRL-TK plasmid for 24 h. The promoter activities were determined as a relative signal of F-luc divided by R-luc.