Fig 2: a The protein levels of FGF15 in serum, ileum and liver tissue by ELISA assays. b the FGF15 mRNA levels and protein levels from 4 groups at month 10. The lysates of ileal tissues from fasting and non-fasting wild type mice are used as positive controls for FGF15, while the lysate of H4IIE cells, a rat hepatoma cell line, is used as a negative control because rats do not express FGF15. N-fasting: non-fasting. c Representative images of FGF15 protein distribution in hepatic parenchyma and computer image-quantification in all 4 experimental groups at month 10 by immunohistochemical staining. Ileal tissues from fasting and non-fasting wild type mice are used as positive controls for FGF15 IHC staining. N-fasting: non-fasting. UT: untreated; CD: control diet; HFD: high fat diet; DEN: N-nitrosodiethylamine. *, P < 0.05 vs UT + CD

Fig 3: a Time-course study of relative mRNA levels of E-Cadherin and vimentin in hepal-1 cells treated by FFA. The representative images of E-Cadherin and vimentin in hepal-1 cells treated by FFA at 72 h. b A trans-well migration assay to study the migratory ability of Hepal-6 cells in response to FFA treatment. Migration cell number was calculated. c A scratch-wound healing assay to study the migratory ability of Hepal-6 cells in response to FFA treatment. A 250 µm scratch-wound was made on 90% confluent cells in each treatment group. Average length of scratch-wound was calculated in each treatment group and the migratory ability of cells was represented as wound closure length. *: P < 0.05 vs 0 h. d Western blot analysis of EpCAM, ß-catenin, FGFR4 and FASN in hepal-1 cells treated by FFA. The protein levels were quantified by image analysis and presented as pixel ratio over the control GAPDH. #: P < 0.05 vs 0 h. FFA: free fatty acid; h: hour. UT: untreated; BLU: BLU9931; XAV: XAV 939. e-f siPPARa and siCD36 as well as the scrambled siRNA were applied to the Hapal-6 and HepG2 cells for 48 h to silence the mRNA of PPAR-a and CD36, and the cells were challenged by FFA for 48 h. Western blot analysis was performed to determine the protein levels of FGF15, FGF19, FGFR4, EpCAM, ß-catenin, andcyclin D. 1: scrambled siRNA; 2: FFA; 3: FFA+ siCD36; 4: FFA+ siPPARa; 5: FFA+ siCD36 + siPPARa. N.S.: no significant importance; *, P < 0.05; **, P < 0.01

Fig 4: a-c Representative images of IHC for FGFR4, ß-klotho, and FASN in hepatic tissues and protein expression by computer image-quantification, and Western blot analysis of protein levels in the 4 experimental groups at month 10. The protein expression by computer image-quantification presented as Average Threshold Area (Average Th Area). The protein levels of Western blot analysis were quantified by image analysis and presented as pixel ratio over the control GAPDH. KLB: ß-klotho; UT: untreated; CD: control diet; HFD: high fat diet; DEN: N-nitrosodiethylamine. *, P < 0.05 vs UT + CD. d mRNA expressions (CD36, PPAR-a and PPAR-?) in the liver tissues from 4 groups at month 6 and month 10. Western blot analysis of FGF15 protein levels in three HCC cell lines (Hepal-6, HepG2 and H4IIE) challenged by FFA 0–72 h. Western blot analysis were quantified by image analysis and presented as pixel ratio over the control GAPDH. e The protein levels of FGF15 and FGF19 in the supernatant of Hepa1–6, H4IIE, and HepG2 cells by ELISA assays. *, P < 0.05; **, P < 0.01