Figure 6

IFNγ-stimulated IRF-1 binds to the IRF-1α sequence of the PD-L1 promoter in EBV (+) GC. (A,B) Chromatin immunoprecipitation (ChIP) assays were performed on IFNγ-stimulated or unstimulated EBV (−) and EBV (+) GC cells using anti-IRF-1 antibody and sequence-specific primers (IRF-1α and IRF-1β) targeting the IRF-1 binding regions of the PD-L1 promoter. After AGS and SNU-719 cells were stimulated with 10 ng/mL IFNγ for 24 h, immunoprecipitated DNA-protein complexes were analyzed by PCR and normalized against input DNA. Anti-H3 antibody and IgG were used as positive and negative controls, respectively. DW, distilled water. (C) Electrophoretic mobility shift assay (EMSA) was performed using nuclear extracts from IFNγ-stimulated or unstimulated AGS and SNU-719 GC cells and biotin-labeled double-strand oligonucleotide probes (IRF-1α and IRF-1β). (D) Competitive EMSA for evaluating the specificity of the IRF-1α binding site was performed on IFNγ-stimulated SNU-719 cells using the biotin-labeled wild-type (Hot), unlabeled wild-type (Cold), unlabeled mutant (Cold mut), and biotin-labeled mutant (Hot mut) IRF-1α probes. (E) Supershift assays for evaluating the binding of IRF-1 at the IRF-1α site were performed using the wild-type IRF-1α probe, IFNγ-stimulated SNU-719 nuclear extracts, and anti-IRF-1 antibody. IgG was used as negative control. The double asterisk and arrowheads indicate a supershifted band and DNA-protein complexes, respectively.