Fig 2: Cholesterol activates Hh signaling in liver CSCs.a Indicated genes of stemness related signaling pathways were analyzed by qRT-PCR in vehicle (DMSO), cholesterol (Chol), and cholesterol biosynthesis inhibitor atorvastatin (Atorv, an HMGCR inhibitor) treated liver CSCs. b Hh signaling genes were analyzed in PPARd depleted and in forced expression of SREBF2 CSCs. Data were normalized to endogenous 18S rRNA expression and sgCtrl were assigned with a value of 1. Data are means ± SD. n = 5. Exact P values from left to right: 0.0059, 0.026, 0.00018, 0.00035, 0.021, 0.032, 0.0037, 0.0045. c Live-cell luminescence traces from liver CSC cells introduced with indicated plasmids. Baseline luminescence was recorded for 10 min, followed by forskolin treatment and continued detecting at 2 min intervals. n = 5. d Liver cancer cells were transfected with Gli-luciferase (Gli-Luc) reporter plasmids, followed by introduced by indicated plasmids. Luciferase activity was measured following treatment with increasing concentrations of Smo agonist SAG21k. n = 5. e Gli-luciferase activity liver cancer cells stimulated with ShhN-conditioned medium in combination with exogenous cholesterol (50 µM) or cholestanol (50 µM) following endogenous sterol depletion by cyclodextrin. n = 6. f Sphere formation assay of cells from e. Scale bar, 100 µm. n = 4 biologically independent samples. g Gli-Luc signaling assay of WT Smo or Smo mutations (V333F or T470Q) at deep 7TM in liver cancer cells stimulated with ShhN-conditioned medium in combination with exogenous cholesterol (50 µM). Data are means ± SD. n = 5. Exact P values from left to right: 0.00092, 0.041, 0.058, 0.14. h Sphere formation assay of cells transfected with WT-Smo or Mut-Smo. Graph data are means ± SD. n = 5 biologically independent samples. Exact P values from left to right: 0.0039, 0.0037, 0.0020. *P < 0.05; **P < 0.01; ***P < 0.001, and NS not significant (P > 0.05) by two-tailed Student’s t-test.

Fig 3: PPARδ promotes liver CSC self-renewal via enhancing cholesterol synthesis.a Heatmap analysis of signature molecules of steroid metabolites under positive ion mode in PPARδ depleted versus sgCtrl treated liver CSC cells. b Heatmap analysis of cholesterol metabolism related genes in PPARδ-depleted and control CSCs. Data were normalized to endogenous 18S rRNA expression and sgCtrl were assigned with a value of 1. c Kaplan–Meier plots of overall survival for SREBF2 in TMA cohort. Median expression value was used as a cut-off. P values for Kaplan–Meier curve were determined using a two-sided log-rank test. d Western blotting analysis of cholesterol metabolism related gene expression in PPARδ depleted, GSK3787 (20 μM) treated and SREBF2 overexpressed liver CSCs compared with control CSCs. n = 3 biologically independent samples. e Levels of total cholesterol, free cholesterol and cholesterol esterin TRMT6/TRMT61A, PPARδ, and SREBF2 depleted liver CSCs. Data are means ± SD. n = 6. Exact P values from left to right: 0.037, 0.0061, 0.0070, 0.0032, 0.046. f Filipin III staining of cellular cholesterol distributions in PPARδ depleted, and co-transfection of SREBF2 in PPARδ depleted liver CSCs from two HCC patients compared with control CSCs (n = 5). Scale bar, 10 μm. n = 3 biologically independent samples. g Total cholesterol levels in liver CSCs treated with vehicle (DMSO), PPARδ antagonist (GSK3787, 20 μM) or agonist (GW501516, 20 μM) for 72 h. Data are means ± SD. Exact P values from left to right: 0.041, 0.0073. CSCs were sorted from HCC patients, n = 15. h, i Representative images of oncosphere formation analysis from liver CSCs treated with vehicle, cholesterol synthesis inhibitor atorvastatin (atorv, HMGCR inhibitor, 5 μM), cholesterol (20, 30, and 40 μM) (upper panel) (h). Oncosphere formation analysis of PPARδ-depleted spheres treated with overexpression of SREBF2, coupling with atorvastatin (atorv) (lower panel) (i). Scale bar, 100 μm. Liver CSCs were sorted from HCC primary samples, n = 10. Data are means ± SD. Exact P values from left to right: 0.0032, 0.047, 0.046, 0.0019, 0.026, 0.038; 0.026, 0.037, 0.0065, 0.0091, 0.043, 0.0029. j Total serum cholesterol levels in 10-week-old normal-fed male MycOE mice transduced with sgPpard using CRISPR/Cas9 strategy for 10 days (Ppard−/−; MycOE). WT:Ppard+/+; MycOE. Data are means ± SD. n = 5 mice per group. Exact P value: 0.0073. These experiments were repeated three times. k Tumor numbers from Ppard+/+; MycOE and Ppard-/-; MycOE mice. Data are shown as means ± SD (n = 6 mice per group). Exact P value: 0.033. *P < 0.05; **P < 0.01 by two-tailed Student’s t-test.

Fig 4: Smoothened deletion in hepatocytes alters hepatic cholesterol homeostasis in chow-fed miceMale Smo (+) and Smo (-) mice were fed a low-cholesterol chow (0.02% w/w) diet for 7 days.(A) Total liver cholesterol.(B) Liver cholesterol esters.(C) Hepatocyte gene expression of SREBP2, HMGCR, LSS, AACS, INSIG1, and ACAT2.(D and E) Western blot and densitometric analysis of hepatocyte membrane and nuclear SREBP2 (precursor, P; active A), EGFR, and TAF15 protein levels.(F and G) Western blot and densitometric analysis of hepatocyte HMGCR, AACS, SRB1, and ß-tubulin protein levels.(H) Hepatocyte gene expression of LXRa, ABCA1, ABCG5, ABCG8, and SRB1. Results reported as mean ± SEM. *p < 0.05, **p < 0.01 vs. Smo (+) chow.See also Figure S1.

Fig 5: Hepatocyte-specific Melanocortin 1 receptor (MC1-R) deficiency enhances cholesterol and triglyceride accumulation in the liver.(A) Representative hematoxylin and eosin (H&E) and Oil Red O-stained liver sections of chow-fed control (Mc1rfl/+ AlbCre/+) and Mc1r LKO mice. Scale bar, 50 µm. (B, C) Quantification of liver total cholesterol and triglyceride content in chow-fed control and Mc1r LKO mice. (D, E) Quantification of plasma total cholesterol and triglyceride concentrations in chow-fed control and Mc1r LKO mice. (F) Representative Western blots of SREBP2, HMGCR, DHCR7, and vinculin (loading control) expression in the liver of chow-fed control and Mc1r LKO mice. (G) Quantification of mature and precursor forms SREBP2 as well as their ratio (precursor-to-mature) in the liver of chow-fed control and Mc1r LKO mice. (H) Quantification of HMGCR and DHCR7 protein levels in the liver of chow-fed control and Mc1r LKO mice. Values are mean ± SEM, n=6–10 mice per group in each graph. *p<0.05 and **p<0.01 versus control mice by Student’s t-test. SREBP2, sterol regulatory element binding protein 2; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; DHCR7, 7-dehydrocholesterol reductase. Figure 2—source data 1.Uncropped Western blots for Figure 2F.