Fig 2: 12-HHTrE-LTB4R2 signaling activates CTNNB1 and YAP1 and auto-induces LTB4R2.a Concentration-dependent suppression of Huh7 cell growth by the LTB4R2 antagonist LY255283. *p < 0.05, **p < 0.01 vs. vehicle control by two-sided t-test (n = 3 separate experiments). b IB analysis of cytosolic and nuclear proteins from Huh7 cells with LTB4R2 KD (LTB4R2-shRNA) vs. control (SCR-shRNA). The numbers shown above the lanes indicate repeated KD experiments (n = 6). c IB analysis for pERK1/2 and ERK1/2 of cytosolic proteins from LTB4R2-shRNA vs. SCR-shRNA Huh7 cells as described above (n = 6 experiments). d IF microscopy for nYAP1 of Huh7 cells cultured in ~40% density and infected with adenovirus expressing EGFP plus LTB4R2-shRNA or SCR-shRNA with morphometric data for the percentage of nuclear YAP1+ cells. NT is a no infection control. Data presented as means ± SEM (n = 3 separate experiments). **p < 0.01 vs. SCR-shRNA cells by two-sided t-test. e TEAD-luciferase activity as the readout of YAP activity in Huh7 cells treated with 12-HHTrE. Data presented as means ± SEM (n = 3 separate experiments). *p < 0.05, **p < 0.001 vs. vehicle treatment (dotted line) by one-way posthoc ANOVA test. f IF microscopy of nuclear YAP1 in Huh7 cells cultured in higher (~65%) density in serum-free media without (NT) or with the treatment of vehicle or 12-HHTrE (50 nM). A bar graph shows morphometric data for the percentage of nYAP1+ cells (n = 3 separate experiments). Data presented as means ± SEM. ***p < 0.001 vs. vehicle by two-sided t-test. g IB analysis of cytosolic and nuclear proteins from Huh7 cells treated with vehicle (Veh.) or 12-HHTrE (50 nM) (n = 3 separate experiments). h 12-HHTrE activation of wild-type vs. TCF site-mutated YAP1 intronic enhancer as measured by luciferase reporter activity. Mutated nucleotides in the enhancer are shown in red. **p < 0.01 and ***p < 0.001 vs. vehicle EtOH by two-sided t-test. Data presented as means ± SEM (n = 3 separate experiments). i ChIP-qPCR data for enrichment of CTNNB1 and RNA polymerase II (RPOLII) at the proximal promoter region (Y2) and the first intronic enhancer region (Y3). *p < 0.05 vs. EtOH by two-sided t test. Data presented as means ± SEM (n = 3 separate experiments). j 12-HHTrE-stimulated LTB4R2 proximal promoter-first intron activities with various deletions as determined by a luciferase reporter in Huh7 cells. **p < 0.01 and ***p < 0.001 vs. vehicle by two-sided t-test. Data presented as means ± SEM (n = 3 separate experiments). For all relevant figures, source data and exact p values are provided in the Source Data file.

Fig 3: aHSC release LTB4R2 ligands in a manner dependent on CYP1B1.a TEAD-luciferase activity in Huh7 cells treated with conditioned medium (CM) from LX2 cells transduced with scrambled shRNA (SCRsh-CM) vs. SCD-shRNA (SCDsh-CM) as compared to the media without Huh7 cells (Media). *p < 0.05 by two-sided t-test. Data presented as means ± SEM (n = 4 separate experiments). b TEAD-luciferase activity in Huh7 cells exposed to SCRsh-CM vs. SCDsh-CM in the presence of the TBXAS1 inhibitor Ozagarel or vehicle DMSO. *p < 0.001 vs. Media, #p < 0.001 vs. DMSO by two-sided t-test. Data presented as means ± SEM (n = 3 experiments). c A scRNA-seq t-SNE plot showing Cyp1b1+ cells and violin plots revealing selective Cyp1b1 expression by Fbln2+ cells. Cell numbers for different cell type groups are provided in Supplementary Data 3. d Contour FACS plots of liver mesenchymal cells isolated from control vs. DEN + WAD treated Rosa26mTmG;Col1a1-Cre; (mTmG;CC) mouse, gated by DAPI (Y-axis) for Vit A fluorescence and FITC (X-axis) for Col1a1-GFP, revealing VitA+GFP- quiescent HSC (blue), VitA+GFP+ aHSC (red), and VitA−GFP+ cells (green). FACS gating strategies are provided in Supplementary Information. e scRNA-seq analysis for expression of 12-HHTrE biosynthetic genes in VitA+GFP+ (top) and VitA−GFP+ (bottom) subpopulations from DEN+WAD mouse (DEN) vs. normal (Cont.) livers. f Violin plots for Cyp1b1 expression by subpopulations based on Lrat, Thy1, and Fbln2 expression in VitA+GFP+ cells from the DEN mouse and g in VitA−GFP+ cells. (See Supplementary Data 3 for parameter values for violin plots and cell numbers for different subpopulations). h CRISPR/Cas9 ablation of CYP1B1 in LX2 cells using the guide RNA-A (sgRNA-A) or -B (sgRNA-B) (top), represses CYP1B1 mRNA. *p < 0.05 vs. control by two-sided t-test. Data presented as means ± SEM (n = 3 separate samples). i Oxylipin concentrations in CM from LX2 cells with CYP1B1 KD are described above. *p < 0.05 vs. control by two-sided t-test. Data presented as means ± SEM (n = 4 separate samples). (Raw data provided in Supplementary Data 4 in Supplementary File). j Reduced stimulatory effects of CM from CYP1B1 ablated LX2 cells on the TEAD promoter activity in Huh7 cells (right). ***p < 0.001 vs. Control CM by two-sided t-test. Data presented as means ± SEM (n = 3 experiments). For all relevant figures, source data and exact p values are provided in the Source Data file.

Fig 4: SCD2 deficiency in aHSC results in global repression in CTNNB1, YAP1, LTB4R2, and its ligands.a A heatmap of DEGs in tumor-adjacent liver (TAL) of Scd2f/f;Col1a1Cre (CC) vs. Scd2f/f mice subjected to the DEN + WAD regimen (n = 3 mice per group). b IB analysis of TAL proteins from Scd2f/f;CC vs. Scd2f/f mice as compared to Scd2f/f control mice. (n = 3 mice per group). c IF microscopy of nYAP1+HNF4A+ hepatocytes, nYAP1+ACTA2+ aHSC, nYAP1+SOX9+ ductular cells in Scd2f/f;CC vs. Scd2f/f mouse TAL. Scale bar = 50 μm. White arrows point the cells with HNF4A+, ACTA2+, or SOX9+ staining while yellow arrows point the cells with the dual staining with nYAP1. d Morphometric analysis of nYAP1+ hepatocytes, aHSC, and ductular cells. *p < 0.05 and **p < 0.01 vs. Scd2f/f mouse TAL by two-sided t-test. Data presented as means ± SEM (n = 3 different sections). Exact p values are shown in the Source Data File. e Co-IF microscopy of nYAP1+HNF4α+ liver tumor cells (HCC). Scale bar = 100 μm. The border between HCC and non-tumorous (NT) areas is indicated by a broken line. Images shown are representative of four pairs of samples analyzed. f Imaging morphometric data for the percentage of nYAP1+HNF4α+ liver tumor cells by 3-dimensional confocal microscopy analysis. *p < 0.05 vs. Scd2f/f by two-sided t-test. Data shown are means ± SEM (n = 4 pairs of samples). g qPCR data for Scd2f/f;CC vs. Scd2f/f mouse TAL (n = 6 each) compared to Scd2f/f control normal liver (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005 vs. Scd2f/f control; #p < 0.05 and ##p < 0.01 vs. Scd2f/f TAL by two-sided t-test. h Lipidomic analysis for PUFA metabolites in Scd2f/f;CC vs. Scd2f/f mouse TAL. Data presented as means ± SEM (n = 3 mouse samples per group). P values determined by two-sided t-test. Red arrows depicting reductions in four specific metabolites in Scd2f/f;CC TAL (Raw data are provided in Supplementary Data File). i IB analysis of LTB4R2 for Scd2f/f;CC (n = 6 mice) vs. Scd2f/f (n = 5 mice) TAL proteins compared to Scd2f/f control mice (n = 3 mice). For all relevant figures, source data and exact p values are provided in the Source Data file.