Journal: Cell Death & Disease

Article Title: The RNA m 6 A reader IGF2BP3 regulates NFAT1/IRF1 axis-mediated anti-tumor activity in gastric cancer

doi: 10.1038/s41419-024-06566-0

Figure Lengend Snippet: 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.

Article Snippet: The antibodies used in present study were: IGF2BP3 (Abcam, ab177477, 1:1000), β-Tubulin (Proteintech, 10094-1-AP, 1:1000), METTL3 (Abcam, ab195352, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:1000), PARP (CST, 9532, 1:1000), Caspase-3 (CST, 14220, 1:1000), Cytochrome c (CST, 4280, 1:1000), IRF1 (CST, 8478, 1:1000), IRF2 (Abcam, ab124744, 1:1000), Histone H3 (Abbkine, ABL1070, 1:1000), STAT1 (CST, 14994, 1:1000), Phospho-STAT1 (CST, 9167, 1:1000), SP1 (Abcam, ab124804, 1:1000), NFAT1 (Abcam, ab92490, 1:1000), eIF4E (Proteintech, 11149-1-AP, 1:1000). β-Tubulin, GAPDH and Histone H3 were used as the loading control for tissues and cells respectively.

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Control, Quantitative RT-PCR, Binding Assay, ChIP-qPCR