Fig. 6.

OASL1 inhibits TBK1-mediated inflammation through regulation of G3BP1 activation in macrophages.

(A) BMMs were isolated from TXNIP^FL/FL mice transfected with p-CRISPR-OASL1 activation or control vector followed by 6 h of LPS (100 ng/ml) stimulation. Western-assisted analysis of OASL1 and G3BP1. (B) Immunofluorescence staining for the G3BP1 expression in LPS-stimulated macrophages after transfecting p-CRISPR-OASL1 activation or control vector (n = 3–4 samples/group). DAPI was used to visualise nuclei. Scale bars, 30 μm. (C) BMMs from TXNIP^M-KO mice were transfected with the p-CRISPR-OASL1 KO or control vector followed by 6 h of LPS stimulation. Western blot analysis of OASL1 and G3BP1. (D) Immunofluorescence staining for the G3BP1 expression in LPS-stimulated macrophages after transfecting p-CRISPR-OASL1 KO or control vector (n = 3–4 samples/group). DAPI was used to visualise nuclei. Scale bars, 30 μm. (E) BMMs from TXNIP^FL/FL mice were transfected with the p-CRISPR-G3BP1 KO or control vector followed by 6 h of LPS stimulation. Western blot analysis and relative density ratio of p-TBK1, TBK1, p-IRF3, IRF3, p-P65, and P65. (F) Quantitative RT-PCR analysis of IL-6, TNF-α, CXCL-10, and IFN-β mRNA levels in LPS-stimulated macrophages after transfecting p-CRISPR-G3BP1 KO or control vector (n = 4 samples/group). All Western blots represent 4 experiments, and the data represent the mean ± SD. Statistical analysis was performed using the permutation t test. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005. BMM, bone marrow-derived macrophage; CRISPR, clustered regularly interspaced short palindromic repeats; CXCL-10, chemokine (C-X-C motif) ligand 10; G3BP1, Ras GTPase-activating protein-binding protein 1; INF-β, interferon-β; IRF3, interferon regulatory factor 3; KO, knockout; LPS, lipopolysaccharide; OASL1, 2′,5′ oligoadenylate synthetase-like 1; TBK1, TANK-binding kinase 1; TNF-α, tumour necrosis factor-alpha; TXNIP, thioredoxin-interacting protein; TXNIP^FL/FL, floxed TXNIP; TXNIP^M-KO, myeloid-specific TXNIP KO.