SUPPL FIGURE LEGENDS:
Supplementary Figure 1: Neutrophil infiltration, proliferation and apoptosis in bile-duct-ligated mice
Neutrophils were visualized by immunofluorescent staining using Ly6G as a primary cellular marker (A). The TUNEL assay was performed 3d after BDL. TUNEL positive Nuclei are shown in green. (B). Liver injury induced cellular proliferation was analysed by staining the ubiquitious expressed cell cycle protein Ki67. All Images were taken in 200-fold magnification, the nuclei were counterstained by DAPI and Ly6G and Ki-67 were visualized by using ALEXA488 (green) conjugated secondary antibodies (C).
Supplementary figure 2: Analysis of CRAMP and pro-inflammatory cytokines in primary hepatocytes and pattern recognition receptor expression after LPS-stimulation
Displayed are data derived from an in vitro experiment, were primary hepatocytes were stimulated with 10µg LPS. Expression of the anti microbial peptide CRAMP (A) was measured by Real-Time PCR. It should be noted, that we observed a clear induction of CRAMP-expression in cultured hepatocytes, despite the absolute expression level in the hepatocyte fraction is lower compared to immune cells.
Here an analysis of the intracellular pathogen related recognition factor NOD1 (B) and of the components of the inflammasome complex NCF4 and Nlrp3 (C, D) obtained 3d after BDL were determined by using Real-Time PCR. Changes in mRNA expression levels were calculated by using GAPDH as a reference gene. *=p<0,05; **=p<0,01; #=p<0,001.
Supplementary figure 3:TLR-signalling after LPS-stimulation of mice
Expression analysis of genes involved in the anti-bacterial defence such as LPS-binding protein (A), CD14 (B), TLR4 (C) , MyD88 (D) and TIRAP (E) as well as TIRAP after BDL (F) both activated by the TLR4 pathway were analysed by Real-Time PCR 6h after stimulation of mice with 10µg LPS. Changes in mRNA expression levels were evaluated against GAPDH as a reference gene. *=p<0,05; **=p<0,01; #=p<0,001
Supplementary figure 4:Analysis of the Acute-Phase-Response after LPS
Western blot analysis was performed to investigate the activation status of STAT3 (A, 1st panel) and JNK1/2 pathway (A 2nd panel and 3rd panel for JNK1/2 activated and inactivated state) after 6h stimulation with 10µg LPS in mice. Relative amounts of protein are compared to GAPDH (A, 4thpanel). Gene expression analysis of the stimulate mice investigating the STAT3 downstream target SAA were made by Real-Time PCR (B). *=p<0,05; **=p<0,01; ***=p<0,005; #=p<0,001