Rsu-1 levels in HCC.

<p>A) Graphical representation of the number of HCC cases positive, negative or moderately positive for Rsu-1 in HCC tissue array (24 cases/48 cores). B) Protein levels of Rsu-1 in HCC cell lines compared to human hepatocytes (HH). Most of the HCC cell lines show decrease in Rsu-1 protein. C) Successful overexpression of Rsu-GFP (fusion protein) in Hep3B cell line. GFP tagged ORF clone of Homo sapiens Rsu-1 (#RG203334, Origene) was transfected into Hep3B cell line and analyzed for Rsu-1 48 h after transfection. Since it is a GFP fused protein, the MW of Rsu-1 is ~fifty-five kd instead of twenty-nine kd (MW of GFP is ~twenty-six kd). D) GFP-Rsu-1 fusion protein associates with PINCH inside the cell. Overexpression of GFP-Rsu-1 in Hep3B cell line leads to association of GFP-Rsu-1 with PINCH. GFP was immunoprecipitated 48 h after transfection. GFP precipitates were probed with either GFP or PINCH. Presence of PINCH in GFP precipitates shows association of GFP-Rsu-1with PINCH.</p

ECM changes in PINCH DKO mice.

<p>A) Photomicrograph of a liver of 5 week old PINCH DKO mouse showing increased stellate cell activation as evident by αSMA stain (shown by arrows). Similar photomicrograph from a Control mouse of the same age shows minimal activation of stellate cells B) Photomicrograph of a liver of a 30 week PINCH DKO mouse showing increased ECM deposition as evident by reticulin stain. All the figures are 100X. The insert are of 200X magnification.</p

Liver regeneration kinetics in PINCH DKO mice.

<p>C) PINCH DKO mice show no termination defect. At Day 14 after partial hepatectomy, the percentage body/liver weight of the control and PINCH DKO mice reaches 100% of the original.</p

Increased hepatocytes proliferation in PINCH DKO mice.

<p>A) Ki67 positive hepatocytes in a 5 and 17 wk old PINCH DKO respectively. B) Ki67 positive hepatocytes in a 5 and 17 wk old control mice respectively.</p

Components of the IPP complex are upregulated at the end of liver regeneration.

<p>Western blot of various components of IPP complex in rat hepatocyte pellet at various time points after partial hepatectomy. Hepatocytes were isolated by 2 step collagenase perfusion.</p

Quantitative assessment of hepatocyte proliferation and apoptosis in PINCH DKO mice.

<p>A) Number of Ki67 positive cells/field at different ages after birth. Each data point is the mean ± SE from two fields per slide from each animal in a total of at least 3 animals. B) Fold change in apoptosis (caspase3/7 activity) in the PINCH DKO cell lysates as compared to the controls at different ages. The numbers were derived as the ratio of caspace 3/7optical density between DKO and control mice. Each data point is the mean ± SE of at least 3 pairs of mice per time point. Comparison between two groups at the same time point is made by unpaired Student’s t test. The criterion for statistical significance is p ≤ 0.05. * indicates statistically significant difference.</p

Morphological changes in PINCH DKO mice.

<p>A) Hepatocytes isolated from PINCH DKO mice show absence of PINCH1 (Note: PINCH2 is already systemically removed). Other components of the IPP complex were also downregulated. Hepatocytes were isolated from 17 week old mice. Western blot shows pooled samples from 3 mice. B) Percent liver weight to body weight ratios of control and PINCH DKO mice at different weeks of age. Each data point is the mean ± SE from more than three measurements per point. Data is expressed as means ± S.E. Comparison between two groups at the same time point is made by unpaired Student’s t test using Microsoft Excel. The criterion for statistical significance is p ≤ 0.05. * indicates statistically significant difference. C) Representative livers of control and PINCH DKO mice at 30 weeks of age indicating the difference in liver size between the two groups. D) Histology of the liver section of the PINCH DKO showing HCC. Representative livers of control and PINCH DKO mice at 1 year of age showing presence of liver tumors.</p

IL-6 synthesis and NFκB signaling in hepatocytes after LPS injection.

<p>(A) FISH for IL-6 mRNAs in serum-free rat hepatocyte cultures, 15 min after media change with 1 µg LPS/ml or diluent (control). For these photographs, as a baseline level of IL-6 mRNA was known to be present (see 1D), gating was adjusted with the diluent-treated sample serving as the baseline. (<b>B, C</b>) Rat livers were injected with 100 µg/kg LPS or saline (control) and harvested at 4 h post treatment. In <b>B</b>, samples were simultaneously stained for albumin protein (green) and IL-6 mRNA (red). Co-localization (merge) appears as yellow. A presumptive inflammatory cell (expressing IL-6 mRNA but albumin-negative) is indicated by an arrow and featured in magnified inserts. Mock hybridizations (not shown) were used as the immunofluorescent gating control. <b>C</b> shows standard immunohistochemical staining using an antibody against IL-6. (<b>D, E</b>) Representative immunohistochemistry depicting nuclear p65 (<b>D</b>), or p50 (<b>E</b>) with and without LPS treatment. Arrows show nuclei stained with p65 or p50 (brown), arrowheads indicate unstained nuclei (colorless). Simultaneously stained tissue section from liver of ILK-null mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096053#pone.0096053-Gkretsi1" target="_blank">[40]</a> shows positive nuclear localization staining for p50 (left panel of <b>D</b>). Dotted boxes are featured in magnified inserts. Scale bars, 20 µm in all images except for <b>D</b> (40 µm).</p

IL-6 in serum-free cultured hepatocytes and Kupffer Cells.

<p>(A) Representative RT-PCR depicting IL-6 from a serum-free rat hepatocyte culture (Heps) at 2 h after attachment (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096053#pone-0096053-g004" target="_blank">Figure 4A</a>) and from fresh Kupffer Cells (KCs) at 15 min after attachment. GAPDH was used as a positive control. Cells are from the same animal. (<b>B</b>) Densitometric analyses depicting percent IL-6 mRNA, as compared to GAPDH, with mean ± s.e.m., in hepatocytes and Kupffer cells. <i>n</i>  =  number of independent trials using separate animals. * indicates statistical significance, <i>P</i> = 0.0238, between hepatocytes and Kupffer cells, two-tailed t-test. (<b>C</b>) Immunofluorescent staining showing IL-6 in rat hepatocytes (top, note the comparatively large size and presence of typical bi-nucleated cells) and Kupffer cells (bottom) plated completely serum-free from the same animal. Secondary antibody was conjugated with Cy3 (red). The left panel is a control visualized at the same gating with no primary antibody added. The right panel is a phase contrast image of the cells taken at the same magnification just prior to staining. (<b>D, E</b>) mRNA FISH depicting IL-6 in hepatocytes (<b>D</b>), or Kupffer cells (<b>E</b>) from the same animal. Arrows indicate IL-6 mRNA-negative Kupffer cells. Animal <i>n</i> = 8. Nuclei were stained with Hoescht dye (blue). Probes were conjugated with Alexa 546 (red). Left to right, hybridization with: reagent control (no probe, used for gating), negative control (labeled IL-6 intron), labeled IL-6 cDNA and labeled GAPDH cDNA (positive control). (<b>F</b>) Representative slot blot samples (bottom) and analyses (top, n = 12) of media from freshly plated hepatocytes at 2 h after attachment (T0) or after another 2 h (2 h). * <i>P</i> = 0.0313 significance, using paired two-tailed t-test. Scale bars, 20 µm in images.</p

NFκB signaling corresponds to changes in IL-6 production in response to HGF.

<p>(A) Confocal staining for the NFκB subunits p50 and p65 in serum-free hepatocyte cultures over 15 min, in the presence or absence of 20 or 500 ng HGF/ml. Actin staining (phalloidin) of the plasma membrane is shown in blue; p50, red; p65, green; with co-localization appearing as yellow. The control cells (no HGF) were used as the immunofluorescent gating control. (<b>B</b>) Summary graph of the percent nuclear staining for NFκB (co-localized staining for p50 and p65) in the experiment shown in A, with mean ± s.e.m. <i>P</i> = 0.0284, significant for one way ANOVA. * indicates statistical significance, <i>P</i><0.05 from control, Newman-Keuls test. (<b>C</b>) Immunofluorescent staining for the NFκB inhibitor, IκB, in serum-free hepatocyte cultures from the experiment shown in A, at 15 min after stimulation with 0 (control), 20 or 500 ng HGF/ml. The cells without primary antibody were used as the immunofluorescent gating control. Scale bars, 20 µm in all images.</p