Suppression of spontaneous proliferation of mouse hepatocytes in the presence of 10

<p>^−<b>6</b><b> M dexamethasone.</b> Tritiated thymidine was added continually in the cultures, from 4 hours after cell plating following perfusion of the liver by collagenase. Cultures were harvested at the indicated time points in the X-axis. Addition of HGF and EGF is indicated as “GFs”. (For growth factor concentration, please see Materials and Methods).</p

Proliferation of mouse hepatocytes maintained in the rat hepatocyte (HGM) medium.

<p>Y-axis indicates incorporation of tritiated thymidine per µg DNA. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p

Interactions between FGF2 and PDGF AA and BB in stimulation of DNA synthesis in mouse hepatocyte cultures.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The growth factors added to the medium are listed under the X-axis.</p

Mouse hepatocyte proliferation in the final formulation of the MHGM medium.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p

Rat hepatocyte proliferation in the MHGM medium.

<p>Y-axis indicates percent of labeled nuclei due to incorporation of bromodeoxyuridine into DNA. The scale of Y-axis is kept identical to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095487#pone-0095487-g004" target="_blank">figure 4</a>, for direct comparison of the responses of mouse and rat hepatocytes. The growth factors added to the medium are listed under the X-axis. CTR: Control cultures, with no growth factors added. Growth factors were added at 24 hours after plating of hepatocytes. D 0–2 (etc.) indicates days in culture after addition of the growth factors. (For growth factor concentration, please see Materials and Methods).</p

Intense vacuolation of hepatocyte cytoplasm in a preliminary formulation of the mouse hepatocyte growth medium (MHGM).

<p>Elimination of nicotinamide removed this cytoplasmic change. A: Phase contrast photomicrograph of the hepatocytes in culture showing intense valuation of the cytoplasm (Magnification: 100X). B: Electron micrograph (5,000X) of hepatocytes carrying vacuoles. There is no apparent connection with bile canaliculi or autophagosomes. C: Oil Red-O stain for lipid demonstrates that the vacuoles observed did not contain lipids.</p

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

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