Fig. 4.

STING increases IRF3 recruitment at Nlrp3 promoter regions by promoting histone methylation via WDR5/DOT1L in AML12 hepatocytes

(A-B) A selected histone-modifying enzyme compound system with 32 targets was applied to test Nlrp3 inhibition in TNFα+Sting-overexpressing AML12 hepatocytes. The heatmap of Nlrp3 expression was analyzed by qPCR (A). Inhibitors that suppress Nlrp3 upregulation are shown (B). (C) IF was used to analyze the colocalization of STING and DOT1L or WDR5 in human cirrhotic livers. (D) AML12 hepatocytes transfected with the Dot1l or Wdr5 overexpression plasmid were treated with vehicle, TNFα (25 ng/mL) for 6 h. The mRNA level of Nlrp3 was analyzed by qPCR. (E) AML12 hepatocytes were treated with DMSO, the DOT1L inhibitor EPZ004777 (7 μM) or the WDR5 inhibitor OICR-9429 (7 μM) for 2 h in addition to either vehicle or TNFα (25 ng/mL)+DMXAA (50 μg/mL) for an additional 6 h. The mRNA level of Nlrp3 was analyzed by qPCR. (F–G) AML12 hepatocytes transfected with the Sting overexpression plasmid were treated with vehicle, TNFα plus OICR-9429 or EPZ004777 for 6 h. Then, cell lysates of nuclear fractionation were subjected to co-IP against p-IRF3, and p-IRF3, WDR5, and DOT1L were then evaluated by WB. (H–J) AML12 hepatocytes transfected with the Sting overexpression plasmid were treated with vehicle, TNFα and TNFα plus OICR-9429 or EPZ004777 for 6 h. Then, p-IRF3 (H), H3K4me2 (I), and H3K79me3 (J) levels at Nlrp3 promoter regions were evaluated by ChIP‒qPCR. n = 3/group. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.