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. 2022 Jul 25;119(31):e2201376119. doi: 10.1073/pnas.2201376119

Fig. 7.

Fig. 7.

IFI16 is a direct target of EZH2 and critical for activation of STING signaling and downstream CXCL10 and CXCL11. (A) ChIP-seq analysis of SKBR3 cells showing H3K27ac and H3K27me3 histone mark enrichment of IFN response gene set near individual transcription start site (−5 kbp to +2 kbp) genome wide. (B) Venn diagram showing the overlapping of H3K27me3hi/H3K27aclow IRG to identify potential direct target of EZH2. (C) ChIP-seq analysis showing H3K27me enrichment and H3K27ac reduction at IFI16, CIITA, and HLA-DRA promoter but not at IRF1, CXCL10, and CXCL11. (D) ChIP-PCR analysis of EZH2 and H3K27me enrichment on IFI16 promoter in SKBR3 cells, using two pairs of ChIP primers flanking the promoter. Data are expressed as means ± SEM of three technical replicates. P values were calculated with one-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, ****P < 0.0001, ns, not significant. (E) ChIP-PCR analysis showing EPZ/ENT abolished H3K27me3 enrichment and induced H3K27ac activation at IFI16 in SKBR3 cells, using two pairs of ChIP primers flanking the promoter. Data are expressed as means ± SEM of three technical replicates. P values were calculated with one way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, ****P < 0.0001, ns, not significant. (F) Knockdown of EZH2 in SKBR3 cells induces IFI16, which can be further enhanced by ENT. (G) EPZ/ENT treatment exhibits synergic effects with NK cells and trastuzumab (Tras) in SKBR3 cells in inducing the expression of IFI16, p-TBK1, p-IRF3, and STING. (H) Knockdown of IFI16 in SKBR3 cells abolished the synergic effects of EPZ/ENT treatment, NK cells and trastuzumab in STING signaling pathway. All Western blot images are representative of two independent experiments. (I) RT-PCR showing knockdown of IFI16 abolished the CXCL10 and CXCL11 induction in SKBR3 cocultured with NK cells and trastuzumab. Data are expressed as means ± SEM of three technical replicates, representative of two independent experiments. P values were calculated two-way ANOVA with Sidak’s multiple comparisons test, ****P < 0.0001, ns, not significant. (J) RT-PCR showing expression of HLA-A, HLA-DRA, and CIITA were not affected by IFI16 knockdown. Data are expressed as means ± SEM of three technical replicates, representative of two independent experiments.