*3.1. LCA Feeding Results in a Significant Upregulation of Genes Associated with Cholestasis, Inflammation, and Extracellular Matrix Remodeling*

Feeding of a chow diet supplemented with 1% LCA over 3.5 days (84 h) resulted in a severe cholestatic liver injury in wildtype (WT) mice, as described previously [21]. Liver tissue of LCA-fed WT mice was compared to chow-fed WT littermates by gene array analysis. In total, 332 and 263 genes were significantly up- or downregulated, respectively (*p* < 0.05; FDR-adjusted, and log2 fold change <−1.5 or >1.5; Supplementary Tables S1 and S2). Overrepresented Gene Ontology (GO) groups among the upregulated genes were associated with proliferation, inflammation, and extracellular matrix organization (Supplementary Tables S1 and S3, Figure 1). The four upregulated genes with the lowest *p*-values were the acute phase proteins serum amyloid A3 (Saa3) (46.2-fold up) and orosomucoid 2 (Orm2) (42.4-fold up), the bacterial siderophore binding protein lipocalin 2 (Lcn2) (32.4-fold up) that is expressed in neutrophils, and the pattern recognition receptor MARCO (macrophage receptor with collagenous structure, 17.4-fold up), which is found on macrophages and dendritic cells (Supplementary Table S1). The most significantly overrepresented GO groups among the downregulated genes were associated with oxidation-reduction processes, sodium-independent organic anion transport, and bile acid biosynthesis. The rate-limiting enzyme of bile acid synthesis, Cyp7a1 (89.0-fold down), was among the strongest downregulated genes (Supplementary Tables S2 and S4). Therefore, gene

expression profiling of the livers of mice under LCA diet revealed a profile of an inflamed, cholestatic organ undergoing extracellular matrix reorganization [21,22].

**Figure 1.** Influence of a chow diet supplemented with 1% lithocholic acid (LCA) over 3.5 days on gene expression in liver tissue of mice. (**A**) Heatmaps of z-scores (expression values standardized per gene to mean 0 and standard deviation 1) of the strongest differentially-regulated genes (*p* < 0.05 and log2 fold change >3 (upregulated) or <−3 (downregulated)). Each row corresponds to a gene, each column to a mouse. Rows and columns were ordered according to average linkage hierarchical clustering. (**B**) Volcano plot: On the x-axis, the log2 fold change is plotted, and on the y-axis, -log10 of the false discovery rate (FDR)-adjusted *p*-value is plotted.

We have previously reported that cholestatic liver injury in response to bile duct ligation is more severe in TGR5 knockout (KO) mice as compared to WT littermates [19]. Therefore, we analyzed liver damage following LCA feeding in TGR5 WT and TGR5 KO littermates.
