Fig 2: Correlation analyses between cGAS–STING signal pathway factors and dysmenorrhea in adenomyosis patients. Correlation analyses between cGAS–STING factors (A) TBK-1 and (B) TNF-a and dysmenorrhea (evaluated by the menstrual VAS pain scores) were performed using Spearman's rank correlation. The coefficient (r) and p values (p) are presented. Evaluation of the menstrual pain VAS pain scores is described in Section Materials and Methods, 2. p < .05 indicates a significant correlation. TBK-1, TANK-binding kinase 1; TNF-a, tumor necrosis factor-a; VAS, visual analogue pain scale

Fig 3: Local cGAS–STING protein expression in the uterine tissue. IHC staining of (A) cGAS and (B) STING in the uterine tissue from the control and adenomyosis (Aden.) patients. cGAS and STING were highly elevated in the adenomyosis patients but not in the control patients. The signals were strongly stained in the ectopic endometrial glands. The quantitative analysis confirmed that cGAS and STING staining was significantly higher in the adenomyosis patients than that in the control patients (A, B, right panels, p < .05, aden. vs. control). Scale bar = 100 µm. The IOD per high-powered field (hpf) was calculated for the IHC-stained images, and the data were presented as the average result of 10 random high-powered fields on the right side in the indicated panel. The Mann–Whitney U test was performed to compare the control and aden. samples, *p < .05. IHC, immunohistochemistry; IOD, integrated optical density

Fig 4: RNF5-deficiency potentiates PRV-induced antiviral immune responses.(A) Immunoblotting analysis of indicated proteins in immunoprecipitated samples and whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA (50 µM). Twenty-four hours after transfection, cells were further transfected with Flag-UL13, Myc-STING, and His-Ub for another 24 h before analysis. (B) Immunoblotting analysis of protein levels of RNF5, UL13, and STING in whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA for 24 h. Cells were then transfected with Flag-UL13 and Myc-STING for the indicated times (above lane). (C) Immunoblotting analysis of RNF5 protein expression in wild type (WT) and RNF5-deficient (RNF5 KO) MEFs. (D) qPCR analysis of transcription of Ifnb1 and downstream genes (Mx1, Isg56) in WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172) and total TBK1 and IKKe, phosphorylated (Ser396) and total IRF3, or RNF5 in whole-cell lysates of WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (F) qPCR analysis of mRNA expression of Ifnb1 and downstream genes (Mx1, Isg56) in RNF5 KO MEFs infected with PRV-WT or PRV-?UL13 (MOI = 1) for indicated times. Data are representative of two (A, C) or three (B, E) independent experiments, or are pooled from three independent experiments (D, F, mean ± SD). *p < 0.05, **p < 0.01 (Student’s t test).

Fig 5: Identification of PRV UL13 as an inhibitor of cGAS-STING signaling.(A) Luciferase activities in UL13 PK-15 or control PK-15 cells cotransfected with IFN-ß-luc reporter or ISRE-luc reporter. Twenty-four hours after transfection, cells were left untreated or stimulated with B-DNA (2 µg/ml) for 12 h, and cell lysates were analyzed for luciferase activity. (B and C) qPCR analysis of IFNB1 and downstream ISGs (MX1, ISG56) mRNA expression in UL13-PK-15 cells and control cells stimulated with B-DNA (1 µg/ml) (B) or cGAMP (1 µg/ml) (C) for the indicated times. (D and E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172)- and total TBK1 and IKKe, phosphorylated (Ser396)- and total IRF3 in whole-cell lysates of UL13-PK-15 cells and control cells stimulated for the various times (above lanes) with B-DNA (1 µg/ml) (D) or cGAMP (1 µg/ml) (E). Data are pooled from three independent experiments (A-C, mean ± SD) or representative of three independent experiments (D, E). *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).