Figure 3.

Metabolic stress induces FGF9 expression in NASH-fib. (a) Hepatic mRNA expression levels of Fgf9 in wild-type mice fed standard diet (normal liver), Col1a2-GFP Tg mice received intraperitoneal CCl₄ injection (CCl₄ liver), and MC4R-KO mice fed WD for 20 weeks (NASH liver) evaluated by quantitative real-time PCR. n = 6. (b) Protein expression levels of FGF9 in normal and NASH livers by Western blot analysis. Blots are shown as cropped images. Uncropped Western blot images are included in Supplementary Fig. S2a. n = 3. *P < 0.05, **P < 0.01 vs. normal liver; ^†P < 0.05 vs. CCl₄ liver. (c) Fgf9 mRNA expression levels in isolated HSCs and activated fibroblasts (CCl₄-Fib, NASH-fib). n = 3. (d) Fgf9 mRNA expression levels in various cell types separated from normal and NASH livers. Resident macrophages, CD45⁺ Ly6G⁻ F4/80^hi CD11b^lo; recruited macrophages, CD45⁺ Ly6G⁻ F4/80^lo CD11b^hi; CD4⁺ T cells, CD45⁺ CD4⁺; and liver sinusoidal endothelial cells (LSEC), CD45⁻ CD146⁺. Hepatocytes were isolated from lean wild-type mice and MC4R-KO mice fed WD for 4 weeks. n = 3–8. *P < 0.05 vs. HSCs; ^†P < 0.05 vs. CCl₄-fib. (e) mRNA expression levels in cultured HSCs treated with TGFβ (10 ng/ml), lipopolysaccharide (LPS, 10 ng/ml), and palmitic acid (200 μM) for 24 hours. (f) Dose-dependent effect of palmitate (100, 200, and 500 μM) on FGF9 induction in HSCs. (g) Effect of various fatty acids (200 μM) on FGF9 induction. Lau, laurate; Ole, oleate. n = 5. *P < 0.05, **P < 0.01 vs. veh; ^##P < 0.01. Data represent mean ± SEM.