Journal: JHEP Reports

Article Title: C/EBPβ–VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

doi: 10.1016/j.jhepr.2025.101418

Figure Lengend Snippet: Presence of hepatic inflammation and changes in hepatic macrophages in the HFHCD-induced MASLD model. (A and B) Representative H&E staining (A) (scale bar = 100 μm) and NAS score (B) based on liver sections. (C) Body weight, liver index (the ratio of liver weight to body weight), and epidymal fat weight at Week 12 (n = 5 for each group). (D) Representative immunofluorescence images showing the distribution of CLEC4F and CD45 in liver sections (scale bar = 50 μm). (E) Flow cytometry analysis of KCs and non-KC myeloid cells from ND and HFHCD mice with relative quantification (n = 4 for the ND group and n = 5 for the HFHCD group). (F) Intracellular flow cytometry analysis showing the protein level of C/EBPβ in KCs (n = 4 for each group). Results were compared by Wilcoxon Rank Sum test (B) or unpaired two-tailed Student’s t-test (D-F). Bars represent mean ± SEM. ∗ p <0.05, ∗∗ p <0.01. C/EBPβ, CCAAT/enhancer binding protein β; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Article Snippet: Cebpb fl/fl mice (Cat. No. T013481) and Clec4f -iCre-2A-tdTomato mice (Cat. No. NM-KI-200069) were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and Shanghai Model Organisms Center (Shanghai, China), respectively.

Techniques: Staining, Immunofluorescence, Flow Cytometry, Quantitative Proteomics, Two Tailed Test, Binding Assay

Journal: JHEP Reports

Article Title: C/EBPβ–VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

doi: 10.1016/j.jhepr.2025.101418

Figure Lengend Snippet: KC-specific Cebpb knockdown alleviates hepatic inflammation in MASLD. (A and B) Representative H&E staining (A) (scale bar = 100 μm) and NAS score (B) based on liver sections (n = 10 for each group from two independent experiments). (C–E) Representative immunofluorescence images of liver sections showing the distribution of CLEC4F and CD45 (C) and CLEC4F and IBA1 (D) (scale bar = 50 μm); the number of CD45 + leukocyte aggregates per 20 × field was additionally counted (E). (F) Relative mRNA levels of EmKC marker genes in livers. (G) Flow cytometry analysis of KCs and non-KC myeloid cells with relative quantification (n = 5 for each group). Results were compared by Wilcoxon Rank Sum test (B, E) or unpaired two-tailed Student’s t-test (F, G). Bars represent mean ± SEM. ∗ p <0.05, ∗∗ p <0.01. EmKC, embryonic KC; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease.

Article Snippet: Cebpb fl/fl mice (Cat. No. T013481) and Clec4f -iCre-2A-tdTomato mice (Cat. No. NM-KI-200069) were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and Shanghai Model Organisms Center (Shanghai, China), respectively.

Techniques: Knockdown, Staining, Immunofluorescence, Marker, Flow Cytometry, Quantitative Proteomics, Two Tailed Test

Journal: JHEP Reports

Article Title: C/EBPβ–VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

doi: 10.1016/j.jhepr.2025.101418

Figure Lengend Snippet: KC-specific Cebpb knockdown reduces hepatic immune cell infiltration by inhibiting leukocyte migration in MASLD. (A–D) RNA-seq was performed on RNA isolated from liver tissue (n = 3 for each group). (A) Principal component analysis. (B) Volcano plot showing upregulated/downregulated DEGs between two groups. (C) Top 10 GO biological processes in GSEA analysis of DEGs. (D) Heatmap showing indicated cell subset marker expression. (E) Relative mRNA levels of hepatic immune cell marker genes in livers. (F–H) Representative immunofluorescence images of liver sections showing the distribution of CD8α (F) and Ly6G (G) with quantification of CD8α + or Ly6G + cells per high-power field (HPF) (H) (scale bar = 50 μm). Results were compared using unpaired two-tailed Student’s t test; n = 7 for the Clec4f -iCre; Cebpb +/+ group and n = 6 for the Clec4f -iCre; Cebpb fl/+ group. Bars represent mean ± SEM. ∗ p <0.05, ∗∗ p <0.01. DEG, differentially expressed gene; GO, gene ontology; GSEA, gene set enrichment analysis; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MoMF, monocyte-derived macrophage; PC, principal component.

Article Snippet: Cebpb fl/fl mice (Cat. No. T013481) and Clec4f -iCre-2A-tdTomato mice (Cat. No. NM-KI-200069) were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and Shanghai Model Organisms Center (Shanghai, China), respectively.

Techniques: Knockdown, Migration, RNA Sequencing, Isolation, Marker, Expressing, Immunofluorescence, Two Tailed Test, Derivative Assay

Journal: JHEP Reports

Article Title: C/EBPβ–VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

doi: 10.1016/j.jhepr.2025.101418

Figure Lengend Snippet: C/EBPβ-mediated transcriptional activation promotes the expression of VCAM1 in KCs of MASLD mice. (A) Activating/repressive function prediction of HFHCD-induced upregulated C/EBPβ peaks in KCs from MASLD mice, with the top five genes in prediction. (B) Expression of indicated genes in scRNA-seq of murine MASLD liver. (C) Relative mRNA levels of indicated genes in the liver. (D) Representative genomic track showing C/EBPβ, ATF3, and p300 distribution at the Vcam1 gene loci in KCs. (E and F) Flow cytometry analysis of the VCAM1 protein level (E) with quantification (F) (n = 3 for each group). (G) Relative mRNA level of Vcam1 in sorted KCs (n = 4 for the “ Clec4f -iCre; Cebpb +/+ HFHCD” group and n = 3 for the other two groups). (H) Relative luciferase activity of the Vcam1 promoter in AML12 cells transfected with murine C/EBPβ overexpression plasmid (left panel, n = 4 for each group) or siRNA targeting C/EBPβ (right panel, n = 3 for each group). Results were compared using an unpaired two-tailed Student’s t test (C, F, and H) or one-way ANOVA and least significant difference (LSD) post hoc test (G). Bars represent mean ± SEM. ∗ p <0.05, ∗∗ p <0.01. C/EBPβ, CCAAT/enhancer binding protein β; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Article Snippet: Cebpb fl/fl mice (Cat. No. T013481) and Clec4f -iCre-2A-tdTomato mice (Cat. No. NM-KI-200069) were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and Shanghai Model Organisms Center (Shanghai, China), respectively.

Techniques: Activation Assay, Expressing, Flow Cytometry, Luciferase, Activity Assay, Transfection, Over Expression, Plasmid Preparation, Two Tailed Test, Binding Assay

Journal: JHEP Reports

Article Title: C/EBPβ–VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

doi: 10.1016/j.jhepr.2025.101418

Figure Lengend Snippet: KC-expressed VCAM1 is upregulated and promotes immune cell infiltration in MASLD. (A, D–F) Wild-type C57BL/6J mice were administered with HFHCD or ND. (A) Relative mRNA levels of Vcam1 in liver tissue. (D and E) Representative immunofluorescence images of liver sections showing the distribution of VCAM1 with CLEC4F and IBA1 (D), and VCAM1 with CD8α (E) (scale bar = 20 μm). (F) Flow cytometry analysis showing the VCAM1 protein level in KCs. (B and C) Vcam1 expression in RNA-seq of liver tissue (B) or FCM-sorted KCs (C) from mice in different dietary MASLD models. (G) Cell adhesion assay in vitro showing the adhesion of RAW264.7 cells to murine primary KCs after administration of murine TNF-α and/or anti-VCAM1 antibody. Results were compared using an unpaired two-tailed Student’s t test (A, F) or one-way ANOVA and an LSD post hoc test (G). Bars represent mean ± SEM. ∗ p <0.05, ∗∗ p <0.01. HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Article Snippet: Cebpb fl/fl mice (Cat. No. T013481) and Clec4f -iCre-2A-tdTomato mice (Cat. No. NM-KI-200069) were purchased from GemPharmatech Co. Ltd. (Nanjing, China) and Shanghai Model Organisms Center (Shanghai, China), respectively.

Techniques: Immunofluorescence, Flow Cytometry, Expressing, RNA Sequencing, Cell Adhesion Assay, In Vitro, Two Tailed Test