Assessment of NK cell activation and hepatic proliferation.

<p>(<b>A</b>) NK cell staining was performed in order to address the role of the immune cell response to HT (blue: DAPI; green: NK1.1(+) cells). This shows, that NK-cells are present in a significantly lower number in HT-recipient mice as compared to controls and solely BM-treated animals. (<b>B</b>) Visualization of cell proliferation by Ki-67 and BrdU incorporation stainings. (blue: DAPI; green: BrdU(+); red: Ki-67(+)). (<b>C</b>) Determination of the total number of BrdU(+) cells by means of BrdU staining in a 100x magnification. (*p<0.05, **p<0.01). (<b>D</b>) Quantification of Ki-67 positive signals in immunofluorescent staining using a 200x magnification. (*p<0.05, **p<0.01). (<b>E</b>) For localization of proliferating cells, double immunofluorescence staining of BrdU and hAAT was performed (blue: DAPI; red: hAAT(+) cells; green: BrdU(+) cells). (<b>F</b>) DAB-staining of NEMO protein in NEMO^Δhepa mice transplanted with Casp8^loxP/loxP/hAAT(+) or Casp8^Δhepa/hAAT(+) donor cells, respectively (DAB in brown: NEMO protein). This clearly shows the presence of NEMO(+) hepatocytes in the surrounding NEMO-deficient (NEMO^Δhepa) liver tissue.</p

Quantitative histological assessment of mouse livers.

<p>Liver histologies were graded for fibrosis levels according to an adapted METAVIR fibrosis score. F0 =  no fibrosis; F1 =  portal fibrosis without septa; F2 =  portal fibrosis with septa; F3 =  numerous septa without cirrhosis; F4 =  cirrhosis.</p

NEMO deficiency in recipient mice favours donor cell selection.

<p>(<b>A</b>) Hepatocyte transplantation was applied to NEMO^Δhepa and WT recipient mice with preceding BMT. Donor cells were derived from WT (Casp8^loxP/loxP/hAAT(+)) or Caspase-8-deficient (Casp8^Δhepa/hAAT(+)) mice, respectively. (<b>B</b>) Serum hAAT levels were analysed via quantitative ELISA and displayed on a logarithmic scale. Basic values represent serum hAAT level 1d post transplantation and are used as a reference value for the calculation of the relative increase in the amount of donor derived hepatocytes over time. (<b>C</b>) Quantitative evaluation of hAAT (+) liver tissue areas 52 weeks after HT. (<b>D</b>) Visualization of engrafting donor cells using hAAT immunofluorescence staining. Clusters of donor derived hepatocytes are displayed 12 and 52 weeks after HT (blue: DAPI; red: hAAT(+) cells).</p

Reduced fibrogenesis in hepatocyte transplanted NEMO^Δhepa mice.

<p>(<b>A</b>) Analysis of Collagen accumulation in transplanted mice via Sirius red staining under polarized light. NEMO^Δhepa mice that underwent transplantation with either Casp8^loxP/loxP/hAAT(+) or Casp8^Δhepa/hAAT(+) donor cells 52 weeks after HT were compared to age-matched completely untreated control mice as well as to bone marrow-transplanted mice 56 weeks (age matched) after BMT. (<b>B</b>) Collagen-1α staining for visualisation of a specific fibrotic collagen subtype (blue: DAPI: red/yellow: Collagen1-α). (<b>C</b>) Assessment of Collagen-1α mRNA expression via quantitative realtime PCR. (**p<0.01, ***p<0.001)</p