Fig 1: Orthotopic HCC development induces a pre-metastatic niche in the lung. (A-B) Pulmonary metastasis was not observed 2 weeks after orthotopic Hepa1-6- implantation or 1 week after orthotopic H22 implantation. (A) Detection of pulmonary metastasis 2 weeks after orthotopic implantation of matrigel or Hepa1-6 cells (upper) and 1 week after orthotopic implantation of matrigel or H22 cells (lower) in the liver by H&E staining. Scale bar, 100 µm. (B) Detection of luc2 by qPCR in the lung of mice 2 weeks after orthotopic implantation of matrigel or Hepa1-6 cells in the liver. (C) Schematic for the procedures of Hepa1-6 and H22 xenograft mouse models. Or., orthotopic implantation. (D-E) Increased numbers of myeloid cells and inflammatory cells in the pre-metastatic lung of orthotopic HCC xenografts. IHC staining (D) and flow cytometry analysis (E) of CD11b+, Ly6G+ and S100A9+ cells in the lung of mice 2 weeks after orthotopic implantation of Hepa1-6 cells in the liver. Scale bar, 25 µm. (F-G) IHC staining (F) and flow cytometry analysis (G) of CD11b+, Ly6G+ and S100A9+ cells in the lung of mice 1 week after orthotopic implantation of H22 cells. Data are displayed as the mean ± SEM; unpaired Student’s t test (B, D, E). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant
Fig 2: Caspase 6 activates NR4A1/SOX9 signaling and induces S100A9 expression in IR-stressed fatty liver.WT and Caspase 6KO mice subjected to fatty liver IR (HIR) were collected to perform RNA-sequencing analysis. A Volcano Plot displaying 615 genes to be upregulated and 440 genes downregulated. B KEGG pathway enrichment analysis of major biological pathways contributing to Caspase 6 function. C Heatmap showing the expression of Top 80 different genes. D, E Western-assisted analysis of NR4A1, SOX9 and S100A9; (F, G) IF analysis of AlexaFluor488-labeled NR4A1 or SOX9 and Cy5-labeled CD68 positive macrophages in ischemic livers, Scale bars, 40 µm, 20 µm; (H) WB-assisted NR4A1 and SOX9 expression profile in Kupffer cells isolated from mice ischemic fatty livers of WT and Caspase 6KO mice. N = 4–7/group, all data represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: S100a9, IL-17, HIF-1a, NF-?B p65, MYD88, TLR4, and TNF-a proteins were expressed in CT26+ mice with ND/DOKD treatment. (a) The representative expression of S100a9, IL-17, HIF-1a, NF-?B p65, MYD88, TLR4, and TNF-a in CT26+ tumor tissues and ß-actin as the internal control for standardization. Quantitative analysis of S100a9 (b), IL-17 (c), HIF-1a (d), NF-?B p65 (e), MyD88 (f), TLR4 (g), TNF-a (h) proteins. Each experiment was repeated three times. All the data were presented as mean ± SEM. Independent-sample T-test was used to compare the data of different groups. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3 per group.
Fig 4: S100A9 is essential for NLRP3 activation and pyroptosis in Caspase 6-mediated inflammation induced by IR in fatty liver.BMMs from WT or Caspase 6KO mice were transfected with the CRISPR-S100A9 Act, CRISPR-S100A9 KO or control vector followed by LPS stimulation; (A, D) Western blotting and relative intensity of S100A9, NEK7, NLRP3 and C-caspase 1 in LPS-stimulated BMMs; (B, E) IF staining for NLRP3 expression in macrophages. DAPI was used to visualize nuclei. Scale bar: 40 µm; (C, F) ELISA analysis of IL-1ß levels in culture medium. N = 4–7/group, all data represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: Knockout of S100A9 ameliorates liver mitochondrial dysfunction through increasing AMPK-medaited glucose and lipid metabolism. (A) Representative images of O2 concentration change (blue line) and O2 flux per mass (red line) in groups. (B) Summarized data for the oxygen consumption capacity measured by high-resolution respirometry in CI leak, CI P (complex I OXPHOS), CI + CII P, CI + CII ETS (electron transfer system capacity), and CII ETS (n = 6). (C) ATP production and (D) fluorescence (represent mitochondrial membrane potential) (n = 6). (E) TEM of liver tissue (left) and the percentage of damaged mitochondria (right, n = 3). Asterisk: normal mitochondria; Arrowheads: damaged mitochondria; N: nucleus; magnification is 2000×. (F) Serum and (G) tissue levels of TG and FFA (n = 6). (H) Immunoblotting analysis of S100A9, p-AKT, AKT, p-AMPK, AMPK, ACC, GLUT4 (left) and quantification of the relative protein levels (right, n = 4). Data are shown as mean ± SEM, and n represents the number of mice in each group.
Supplier Page from Abcam for Anti-S100A9 antibody [EPR22332-75]