Fig. 6

CX3CR1/MYD88/NF-κB regulated the expression of S100A4 in MAFLD progression. a Violin plots showing the expression of Toll-like receptors (TLRs) and adaptor MYD88 across each myeloid cell cluster. b, c Immunofluorescence analysis showing the distributions of CX3CR1⁺CCR2⁺MYD88⁺ cells in the KO and WT mice, HFD-fed and control diet-fed mice (n = 3, scar bar = 10 μm). d Immunohistochemical staining analysis of MYD88 in WT and KO mice (n = 3, scar bar = 50 μm). e Immunofluorescence analysis showing the colocalization of CX3CR1 and MYD88 in PMA-treated THP-1 cells by confocal fluorescence microscopy (scar bar = 20 μm). f Co-Immunoprecipitation analysis showing interaction between CX3CR1 and MYD88 in PMA-treated THP-1 cells. g Western blot showing the expression of NF-κB family proteins and S100A4 after treated with CX3CR1 inhibitor JMS-17-2. h Schematic diagram showing the procedures of RELA (P65) binding to the promoter of S100A4 and subsequent assays. i CUT-RUN analysis showing the binding capacity of RELA (p65) on three binding sites of S100A4 promoter regions in PMA-treated THP-1 cells. j Relative luciferase activity of mutant plasmid and wild-type plasmid of RELA binding sequences (site 3 in h) in 293T cells. k QRT-PCR analysis indicating mRNA expression of S100A4 after treated with CX3CR1 inhibitor JMS-17-2 in PMA-treated THP-1 cells. l Western blot showing the expression of NF-κB family proteins and S100A4 after treated with Diprovocim (NF-κB activator) or JSH-23 (NF-κB inhibitor). m QRT-PCR analysis showing S100A4 mRNA expression after treated with NF-κB activator/inhibitor and MyD88 inhibitor (ST2825) in PMA-treated THP-1 cells. n Oil red O staining and ROS analysis of HUH7 cells with HF treatment (12 μmol/l oleic acid and 6 μmol/l palmitic acid) and control solution for 48 h (scar bar = 100 μm). o QRT-PCR analysis showing the expression of S100A4 of PMA-treated THP-1 cells after cultured by HF conditional medium for 24 h. p Rescue assay showing the expression of S100A4 in PMA-treated THP-1 cells after cultured by HF conditional medium with/without CX3CR1 inhibitor, MyD88 inhibitor or NF-κB inhibitor. q QRT-PCR analysis showing the expression of HSC activation markers such as ACTA2, COL1A1, and TGFB1 of LX2 cells with/without high-fat supernatant stimulation as well as cocultivation with monocyte-derived macrophages (Mo-macs). Data are represented as mean ± SEM. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001