Fig 1: ELISA-based verification of the candidate signatures regulated by HYQ against viral pneumonia in mouse blood. Each group contained 6 mice, and the results from each mouse were analyzed by one-way analysis of variance; 3 and 6 days: virus infection time. ns: no significance. Lgals3bp, galectin-3-binding protein; Serping1, plasma protease C1 inhibitor; Gpx3, glutathione peroxidase 3; Hpx, hemopexin; S100a8, protein S100-A8.
Fig 2: MANF deficiency in myeloid cells activates S100A8/A9-TLR4 signal pathway in mice with liver fibrosis. (A) MANF deficiency in myeloid cells upregulated hepatic S100A8, S100A9, TLR4 and p-p65 levels detected by using immunohistochemistry assay. (B) The quantitative data in panel A. (C) S100A8, S100A9, TLR4, p-p65 and p65 were detected by Western blot. (D) The quantitative data in panel C. (E) S100a8, S100a9, and Tlr4 mRNA levels were detected by qPCR. (F) MANF deficiency in myeloid cells increased serum S100A8 and S100A9 levels. Serum S100A8 and S100A9 levels were detected by ELISA. Data are expressed as mean ± SEM, n = 10; ∗∗P < 0.01, ∗∗∗P < 0.001. WT, wild type; HKO, MANF knockout in hepatocytes; MKO, MANF knockout in myeloid cells; CCl4, carbon tetrachloride.
Fig 3: MANF interacts with S100A8 to impede S100A8/A9-TLR4 signaling. (A) Interaction of MANF and S100A8 was detected by Co-IP. (B) Colocalization of MANF (red) and S100A8 (green) was detected by immunofluorescent staining. DAPI (blue) was used to stain nuclei. (C, D) MANF overexpression inhibited intracellular (C) and extracellular (D) interaction of S100A9 and S100A8 detected by Co-IP with anti-S100A8. (E, F) MANF deficiency in macrophages increased colocalization of S100A9 and TLR4 in peritoneal macrophages (E) and hepatic macrophages (F) by detecting TLR4 (green) and S100A9 (red), n = 5. DAPI (blue) was used to stain nuclei. WT, wild type; MKO, MANF knockout in myeloid cells; LPS, lipopolysaccharide.
Fig 4: USP50 stimulates HMGB1 release in DGR-induced gastric inflammation. (A, B) mRNA levels of HMGB1, HSP70, HSP90, S100A8, and S100A9 were detected via qRT-PCR in U937 cells (A) or RAW264.7 cells (B) treated with DMSO or TDCA (200μM, 24 h). (C-H) ELISA was used to measure the concentrations of HMGB1 (C), HSP70 (D), HSP90 (E), S100A8 (F), and S100A9 (G) in the supernatant of U937 cells and RAW264.7 cells, while ATP concentration (H) was surveyed by ATP assay kit. (I) The protein level of HMGB1 was detected in gastric tissues of Sham and GJ mice by western blotting. Image J was used for quantitative analysis, with GAPDH as loading control. (J) The level of HMGB1 between normal gastric tissues and gastric cancer tissues was analyzed via GEPIA database (gepia.cancer-pku.cn/). (K) The IHC staining of HMGB1 was performed with sections from BRG patients and GC patients. (L) IRS was calculated for (K). (M, N) HMGB1 was assayed by ELISA in the supernatant of U937 cells with USP50 overexpression (M) or knock-down (N). *P < 0.05, **P < 0.01, and *** P<0.001. "ns" means "no significance" (P>0.05).
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