Fig. 2

EZH2-mediated H3K27me3 on the CD274 promoter controls PD-L1 expression. a Hep3B cells were pretreated with GSK126, DZNep, or DMSO for 48 h, and then treated with IFNγ for an additional 24 h. Immunoblotting was performed to detect the protein expression of PD-L1, EZH2, and H3K27me3. GAPDH and H3 were used as loading controls. b An H3K27me3 ChIP assay was performed in shEZH2 Hep3B and vector control cells. H3K27me3 levels at the CD274 (PD-L1) promoter were normalized to the input. TSS, transcription start site, − 0.3, − 0.5, − 1.0, and − 1.5 kb indicate the corresponding upstream loci of the CD274 gene TSS. CXCL10 was used as a positive control. (Mean ± S.E.M.; n = 3; * P < 0.05, ** P < 0.01, Wilcoxon test). c Diagram of the CpG island distribution on − 2000 nt to + 250 nt region of the CD274 promoter predicted by MethPrimer website. d DNA methylation on the CD274 promoter. DNA methylation at CpG sites was quantified using bisulfite sequencing. Filled circle, methylated; open circle, unmethylated. e DNA methylation and gene expression data for PD-L1 from TCGA HCC tissues were analyzed on the cBioportal website. The Pearson correlation coefficient (r) is shown. f and g Effect of downregulation of EZH2 on the IFNγ-STAT1 signaling activation. Huh7 (f) or PLC/PRF/5 (g) cells were pre-transfected with EZH2-targeted siRNA or NC for 48 h, and then treated with IFNγ for 0–4 h. Immunoblotting was performed to detect the levels of pSTAT1 and EZH2. STAT1 and GAPDH were used as loading controls for pSTAT1 and EZH2 respectively