Fig 2: LXRa activation antagonizes TGFß-induced Snail expression a Snu449 cells were transfected with 30 nM of non-targeting siRNA control or LXRa targeting siRNAs and stimulated by TGFß1 (5 ng/ml) for 24 h in starvation medium. Expression of the indicated genes was assessed by real-time PCR. Mean ± SD values are plotted, and basal expression levels set to 1 correspond to the same absolute levels of SNAI1 mRNA expression. Experiments performed in biological duplicate, each of them in technical triplicate. b Snu449 were transfected with 1 µg of pCDNA3 or pCMX-LXRa plasmid, and treated with TGFß1 (5 ng/ml) for 24 h in starvation medium. Expression of the indicated proteins was assessed by immunoblotting. Experiments performed in biological triplicate. Densitometric quantification is provided graphically, and basal expression levels set to 1 correspond to different absolute levels of protein expression, as visualized on the immunoblots. c Snu449 cells were treated with TGFß1 (5 ng/ml) with or without T0901317 (5 µM) in starvation medium for 24 h; expression of SNAI1 mRNA was assessed by real-time PCR. Mean ± SD values are plotted. Experiments performed in biological triplicate, each of them in technical triplicate. d Snu449 cells were treated in starvation medium for 24 h; TGFß1 (5 ng/ml) was administered with or without LXR agonist (T0901317 or GW3965, 5 µM). Expression of Snail was determined by immunoblotting. Experiments performed in biological quadruplicate. Densitometric quantification is provided graphically, and basal expression level set to 1 corresponds to almost undetectable absolute level of Snail expression, as visualized on the immunoblot. e Snu449 cells were immunostained for Snail and DAPI after 3 h of the indicated treatments. Snail is represented in green, DAPI staining in blue. Bars represent 40 µm. Quantification of the percent of Snail-positive nuclei is graphed as mean ± SD values. Experiments performed in biological triplicate. f Snu449 cells were starved and treated as specified. Nuclear fractionation assay was performed and abundance of the indicated proteins was assessed by immunoblotting. Experiments performed in biological duplicate. Densitometric quantification is provided graphically, and basal expression levels set to 1 correspond to the cytoplasmic levels of Snail protein expression, as visualized on the immunoblot. Statistical significance was assessed by two-way (a, b) or one-way (c–f) Anova. Significance was assigned as *p < 0.05, **p < 0.01, ***p < 0.001. All immunoblots indicate molecular size markers in kDa

Fig 3: TGFß regulates LXRa expression in the mesenchymal cell line Snu449 a Hep3B (left) and Snu449 (right) cells were treated with the indicated compounds (10 µM) and TGFß1 (5 ng/ml) for 24 h. The TGFß type I receptor kinase inhibitor LY2157299 was added at a final concentration of 2 µM for 24 h as a potent positive control. The expression of the indicated proteins was analyzed by immunoblotting. Experiments performed in biological duplicate. Densitometric quantification is provided below each lane. Results indicated as <0.01 represent a densitometric intensity between 0.0045 and 0.0082; results indicated as <1 × 10-4 mean a densitometric intensity between 0.00002 and 0.00009; basal expression levels set as 1 correspond to either strong detectable protein levels (E-cadherin) or very low to almost undetectable levels (fibronectin). b, c Hep3B (b) and Snu449 (c) cells were treated for different time periods with TGFß1 (5 ng/ml). The expression of LXRa, LXRß, and SERPINE1/PAI1 genes was assessed via real-time PCR and normalized to the expression of GAPDH. Mean ± SD values are plotted, and basal expression levels set to 1 correspond to different absolute levels of expression for each mRNA. Experiments performed in biological triplicate, each of them in technical triplicate. d Snu449 cells were treated with TGFß1 (5 ng/ml) for the indicated time periods. The expression of the indicated proteins was analyzed by immunoblotting. Experiments performed in biological triplicate. Densitometric quantification is provided graphically, and basal expression levels set to 1 correspond to different absolute levels of expression for each LXR member. e Snu449 cells were treated with vehicle or TGFß1 (5 ng/ml) for 24 h. SB203580 (p38 inhibitor, 10 µM), SP600125 (JNK inhibitor, 10 µM), and PD184352 (MEK inhibitor, 0.5 µM) were added 1 h prior to TGFß1 addition. The expression of the indicated proteins was visualized via immunoblotting. Experiments performed in biological triplicate. Densitometric quantification is provided graphically. Statistical significance was assessed by one-way (b–d) or two-way (e) Anova. Significance was assigned as *p < 0.05, **p < 0.01, ***p < 0.001. All immunoblots indicate molecular size markers in kDa

Fig 4: IL-1ß induced NF-?B signaling suppresses LXR and ABCG5/8. a Liver mRNA for Abcg5, Abcg8, and Nr1h3 in WT chow mice relative to WT DSS/PN, WT PN-only, Ccr2-/-DSS/PN, Il1r-/- DSS/PN, and Anakinra DSS/PN mice. b, c Liver mRNA for Abcg5 and Abcg8 in WT mice left untreated, or IL-1ß i.p. injected (4 h) (upper panels) and in mice LPS i.p. injected (4 h), with or without prior (16 h) i.p. clodronate-liposome (Clo-lip) injection (bottom panels). *p < 0.05 vs. all other groups using t test (upper panels) or one-way ANOVA and Tukey's correction (lower panels). c Liver mRNA for Abcg5 and Abcg8 in WT mice left untreated, or i.p. LPS injected relative to that Casp1/11-/- and Il1r-/- mice. d Liver mRNA for Abcg5, Abcg8, and Nr1h3 in untreated mouse primary hepatocytes relative to LXR agonist (GW3965, 2 µM, 16 h) and IL-1ß (additional 4 h) treatment. e qPCR from LXRß binding site within the ABCG5/8 intergenic region and ChIP for LXRa or NF-?B p65 and p50 on liver homogenate from WT chow, WT DSS/PN, and Il1r-/- DSS/PN mice. Data represented as fold change over IgG; POLII shown as control. f ChIP from HuH7 cells left untreated (HuH7), or exposed to LXR agonist (GW3965, 2 µM; HuH7 + GW3965) for 16 h with or without additional exposure to IL-1ß for 4 h. Specific antibodies for either LXR (left) binding to LXR promoter (top left) or ABCG8 intergenic region (bottom left) or NF-?B p50 and p65 (right) binding to the LXR promoter (top right) or ABCG8 intergenic region (bottom right). g Relative mRNA in primary mouse hepatocytes for Nr1h3, Abcg5, and Abcg8 exposed to IL-1ß (2 ng/ml) with or without prior exposure (1 h) to NF-?B inhibitor (BAY-11-7082, 50 µM. h mRNA for ABCC2 in HepG2 cells exposed to transfection reagent (vehicle) or control siRNA and ABCG8 siRNA and exposure to FXR agonist GW4064 (5µM) with and without stigmasterol acetate (20 µM) (d, g, h depict mean ± SEM of technical triplicates from one representative out of at least five experiments). a, d, g *p < 0.05 vs. all other treatments using one-way ANOVA and Tukey's correction

Fig 5: Hypothetical model of PNAC pathogenesis. Increased intestinal permeability secondary to intestinal inflammation and dysbiosis promotes increased absorption of LPS into portal vein with subsequent TLR4-mediated activation25 and caspase-1- dependent generation of IL-1ß in recruited CCR2+ macrophages (phytosterols contribute to macrophage activation and IL-1ß synthesis)26. Through IL-1 receptor (IL-1R) binding on hepatocytes, IL-1ß subsequently activates NF-?B in hepatocytes, which interferes with the ability of LXR and FXR to bind to promoters (red crosses) and activate expression of ABCG5/8, ABCB11, and ABCC2, resulting in decreased expression of BSEP, MRP2, and ABCG5/8 (blue downward arrows) and phytosterol retention (green dots). Retained phytosterols antagonize FXR activity leading to additional reduction in BSEP and MRP2 expression and subsequent retention of bile acids and bilirubin (additional phytosterol effects indicated by green downward arrows) and cholestasis. In addition, direct effects of LPS on the hepatocyte can suppress FXR activity (dotted line from LPS to hepatocyte)