Fig. S1.LCN2 expression in cultured HSC/MFB. (A) A semi-quantitative RT-PCR was performed to analyze the mRNA expression of LCN2 in culture-activated HSC and transdifferentiated MFB that were cultured for indicated days. To demonstrate ongoing transdifferentiation, the expression of α-SMA was analyzed in parallel. The expression of GAPDH was taken to demonstrate the integrity of cDNA samples. All primers used in this analysis are listed in Table 1. (B) Protein extracts were prepared from HSC that were cultured for indicated times. Equal protein amounts (15 μg/lane) were then subjected to Western blot and subsequently analyzed for expression of fibronectin, collagen type I, α-SMA, LCN2, rS6, and β-actin. The membrane was stained with Ponceau S to demonstrate equal protein loading.

Fig. S2. Immunocytochemical analysis of LCN2 expression in HSC. (A) Primary HSC were isolated and cultured for indicated times (1 or 2 days) on cover slips. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized using 3% Triton X-100 in 10 mM sodium citrate buffer for 2 min on ice followed by incubation with a polyclonal goat anti-rat LCN2 antibody or a rabbit anti-human antibody specific for the GFAP representing a marker of HSC. After washing, the cells were next incubated with biotinylated secondary antibodies followed by avidin-conjugated peroxidase and 3,30-diaminobenzidine substrate, while nuclei were briefly counterstained with hematoxyline. For specificity we used goat or rabbit IgG as a control. (B) In this analysis, the cells were treated as described in (A), followed by streptavidine FITC-conjugated substrate and nuclei were counterstained with DAPI. Slides were analyzed for fluorescence signals under a UV microscope.

Fig. S3. LCN2 expression in chronic CCl4 induced liver injury. Western Blot of CCl4 rat livers show rapid induction and sustained LCN2 expression throughout 12 week-experimentation. The increase of LCN2 expression correlated with the expression of collagen type I and α-SMA. β-actin and rS6 served as loading controls in this analysis.

Fig. S4. Quantitative mRNA expression analysis of LCN2, Vimentin, PAI-1 and fibronectin in cultured hepatocytes. RNA from primary hepatocytes that were cultured for indicated times were isolated and analyzed by quantitative real time PCR for expression of (A)LCN2 or (B) three genes (Vimentin, PAI-1, and Fibronectin) that are engaged in the process of EMT.

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