Fig. 4

EZH2 inhibits PD-L1 transcription by inhibiting transcription factor IRF1. a Huh7 cells were transfected with NC or EZH2-targeted siRNAs for 72 h, and then treated with IFNγ for the indicated times. Immunoblotting analysis was performed to detect the levels of EZH2, IRF1, and PD-L1. GAPDH was used as a loading control. b Huh7 and Hep3B cells were transfected with NC or EZH2-targeted siRNAs, with or without IRF1-targeted siRNAs for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2 and PD-L1. c After transfection with EZH2 siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2, IRF-1 and PD-L1. d After transfection with the indicated siRNA targeting 3′-UTR, Huh7 and Hep3B cells were transfected with the indicated plasmids for 48 h, and then treated with IFNγ for 24 h. Immunoblotting was performed to detect the levels of EZH2, IRF-1 and PD-L1. In c and d, the corresponding control groups were transfected with NC siRNA or vector plasmids. pEZH2 and pIRF-1 represent ectopic expression of EZH2 and IRF-1, respectively. e Huh7 and PLC/PRF/5 cells were pretreated with GSK126, DZNep, or DMSO for 48 h, and then treated with IFNγ for an additional 12 h. GAPDH and H3 were used as loading controls for EZH2 and H3K27me3, respectively. f An H3K27me3 ChIP assay was performed in shEZH2 Hep3B and vector control cells. H3K27me3 levels on the IRF1 gene promoter were normalized to the input. TSS, transcription start site; − 0.5 kb, − 1.0 kb, − 1.5 kb indicate the corresponding upstream locus in the IRF1 gene TSS. CXCL10 was used as positive control (Mean ± S.E.M.; n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001, Wilcoxon test)