Fig 2: TMAO and TNF-a alter expression of the DEGs in Hepa1-6 cells and Huh7 cells. (A–D) Expression of the POSTN, NAPB, LAYN and HTRA3 mRNAs were significantly upregulated in Hepa1-6 cells and Huh7 cells treated with 50 ng/ml TNF-a and 50 µM TMAO in combination compared to cells treated with 50 ng/ml TNF-a alone; results are mean ± SD (n = 3 per group); Student’s t-test. (E,F) On the contrary, 50 ng/ml TNF-a and 50 µM TMAO in combination significantly downregulated the expression of AANAT and AFM compared to cells treated with 50 ng/ml TNF-a alone; results are mean ± SD (n = 3 per group); Student’s t-test.

Fig 3: Single-cell network analysis identifies cardiac fibroblast Htra3.a Isolation of non-cardiomyocytes and fibroblasts for scRNA-seq using fluorescence-activated cell sorting (FACS). Sorted live non-cardiomyocytes (non-CMs) were used for scRNA-seq using the 10X Genomics platform. Sorted fibroblasts with an antibody against PDGFR-a were used for scRNA-seq using the full-length Smart-seq2. b, c Ligand-receptor interaction map in the heart. Genes of ligands and receptors were projected on a two-dimensional map based on their correlation structure. Cell types were annotated by cell-type-specific expression profiles of genes assigned to each module. Edges indicate correlation (b) or interaction (c). d Pathway analysis of genes illustrated on the interaction map in the heart. p-values are determined by Fisher’s Exact test. e Heatmap showing the number of interactions between cell types in the heart. VSMC, vascular smooth muscle cells. f Gene ontology (GO) analysis of upregulated genes after pressure overload in cardiac fibroblasts. Representative genes are also shown. p-values are determined by Fisher’s Exact test. g Co-expression network of cardiac fibroblasts. h Bar graph showing the top 10 genes most correlated with the cardiac fibroblast module. i Uniform Manifold Approximation and Projection (UMAP) plot of single-cell transcriptomes of non-cardiomyocytes (n = 1783) from the murine heart (n = 2). Cell-type annotation (left) and Htra3 expression (right) are on the UMAP plot. j RNA in situ hybridization of Htra3 and immunostaining of Pdgfr-a using the paired mirror cardiac sections. Arrows indicate the colocalization of the Htra3 and Pdgfr-a in the same cells. k RNA in situ hybridization of HTRA3 in the human heart. Arrows indicate HTRA3 expression.

Fig 4: Correlations between the DEGs and immune cell infiltration in liver hepatocellular carcinoma (LIHC). (A–C) The expression levels of POSTN, HTRA3 and AANAT significantly positively correlated with the levels of neutrophil and Treg infiltration and negatively correlated with the levels of activated NK cell and CD8+ T cell infiltration. (D,E) The expression levels of NAPB and LAYN significantly positively correlated with neutrophil, Treg, activated NK cell and CD8+ T cell infiltration. (F) The expression levels of AFM negatively correlated with the levels of neutrophil, Treg and activated NK cell infiltration.

Fig 5: Associations between the DEGs and patient outcomes in liver cancer. Overall survival curve for patients with liver cancer in the GEPIA database (right) and overall survival and disease specific survival curves (left, center) for patients with liver cancer in the Kaplan-Meier plotter databases stratified by expression of (A) POSTN, (B) LAYN, (C) HTRA3, (D) AANAT, (E) AFM, and (F) NAPB.