Fig 2: Pellino2 is required for IFN-β secretion in response to CpG in DCs. (A) WT and Peli2−/− BMDCs were treated with 1 μg/ml CpG ODN 1826. After 24 h, supernatants were tested for IFN-β by ELISA. (B) WT and Peli2−/− BMDCs were treated with 1 μg/ml CpG ODN 1826. After 3 h, Ifnb mRNA levels were determined in cells by RT-PCR. (C) WT and Peli2−/− BMDCs were treated with lipofectamine alone and transfected with 10 µg/ml polydA:dT and 10 µg/ml VACV70 or 10 µg/ml cGAMP alone. After 24 h, supernatants were tested for IFN-β by ELISA. (D) Immunoblot and densitometry analysis of total IRF1, IRF3, and IRF7 in lysates of WT and Peli2−/− BMDCs stimulated with 1 µg/ml CpG ODN 1826 for indicated durations. β-actin was used as a loading control. (A and C) Results are representative of three independent experiments. Data show means + SD for three technical replicates. (B) Results show mean of three independent experiments. Data show means + SEM. (D) Immunoblots show representative of three independent experiments. Data show means + SEM of three independent experiments. WT BMDC versus Peli2−/− BMDC. **p < 0.01, ****p < 0.0001, two-way ANOVA.

Fig 3: PICH deficiency induces type‐I IFN production. A) WT and Pich‐KO LSK cells were subjected to gene expression profiling through RNA sequencing. The enriched IFN‐β response gene set is shown. B) WT and Pich‐KO FL cells were subjected to mass spectrometry analysis, showing expression of BST2. C) mRNA expression of type‐I IFN genes in Pich f/f (Pich flox/flox) and Pich Δ/Δ (conditional knock out of Pich in hematopoietic cells using vav‐cre) fetal livers, as analyzed by RT‐PCR, n = 3 for each, n = 3 for each. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. D) IFN‐β levels were determined by FACS in LSKs of Pich f/f and Pich Δ/Δ mice, n = 7 for each. ∗∗∗p ≤ 0.001. E) Protein levels of IFIT1, IRF3, and IRF7 were determined by western blotting in Pich f/f and PichΔ/Δ FLs.

Fig 4: Macaca mulata TRIM4 overexpression activates the interferon (IFN) pathway. (A) The TRIM4 plasmid was transfected into MARC145 cells. After 48 h, western blotting was performed with the indicated antibody. (B) After TRIM4 transfection in MARC145 cells, RNA was extracted 24 h post-transfection. At the same time, a poly(I:C) (1.0 μg/mL) control group was set up, 1.0 μg/mL poly(I:C) treated MARC145 cells, cellular RNA was extracted at 9 h post-transfection. The mRNA levels of IFN-alpha, IFN-beta, RIG-I, MAVS, IRF3, IRF7, OAS1, IFIT3, and CCL5 were determined by qPCR. (C) Overexpression of Macaca mulata TRIM4 promoted the activities of IFN-beta, NF-κB, and ISRE promoters. The TRIM4 plasmid (300 ng), 200 ng of IFN-beta reporter plasmid, NF-κB reporter plasmid, ISRE reporter plasmid, and 50 ng of ng RL-TK plasmid were co-transfected into MARC145 cells in a 24-well plate, and the activity of these reporters was detected at 48 h post-transfection. (D) Overexpression of TRIM4 increases the phosphorylated levels of interferon-related molecules. The TRIM4 plasmid (500 ng) were transfected into MARC145 cells in a 24-well plate, after 24 h post-transfection, 1.0 μg/mL poly(I:C) was transfected, cell lysates were blotted with the indicated antibodies. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: PICH deficiency promotes cGAS‐STING activation. A) WT and Pich‐KO LSK cells were subjected to gene expression profiling through RNA sequencing. The enriched cGAS‐STING pathway gene set is shown. B) Protein levels of PICH, cGAS, and STING were determined by western blotting in Pich f/f, Pich Δ/Δ, cGAS Δ/Δ (conditional knock out of cGAS in hematopoietic cells using vav‐cre), and cGAS Δ/Δ Pich Δ/Δ (conditional double knock out of cGAS and Pich in hematopoietic cells using vav‐cre) FLs. C) Protein levels of IFIT1, IRF7, and IRF3 were determined by western blotting in Pich f/f, Pich Δ/Δ, cGAS Δ/Δ, and cGAS Δ/Δ Pich Δ/Δ FLs. D) mRNA expressions of type‐I IFN genes in Pich f/f, Pich Δ/Δ, cGAS Δ/Δ, and cGAS Δ/Δ Pich Δ/Δ fetal livers, as analyzed by RT‐PCR, n = 3 for each. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. E) Immunofluorescence analysis of dsDNAs in LSK cells isolated from E14.5 fetal livers. A representative immunofluorescence image is shown (Scale bar = 10 µm). 100 cells per group. ∗∗∗p ≤ 0.001.