1. Volume 125, Issue 1, Pages 192-201 (July 2003)
Protection against liver damage by cardiotrophin-1: a hepatocyte survival factor up- regulated in the regenerating liver in rats 
Matilde Bustos, Naiara Beraza, Juan-Jose Lasarte, Elena Baixeras, Pilar Alzuguren, Thierry Bordet, Jesus Prieto 
Gastroenterology 
Volume 125, Issue 1, Pages (July 2003)
DOI: /S (03)00698-X

2. Figure 1
Effects of CT-1 on H35 cells. (A ) Analysis by flow cytometry of cell cycle and annexin V binding of H35 cells cultured without serum in the presence or in the absence of 50 ng/mL of CT-1 for 7 days. Representative experiment run 4 times. Percentages correspond to hypodiploid cells (left panels) or to annexin V-positive cells (right panels). Apoptosis is associated with increased hypodiploid peak (as a result of nuclear fragmentation) and with increased annexin V binding (as a result of phosphatidyl-serine exposure on the outer layer of the cell membrane). The mean values ± SEM of the proportion of hypodiploid cells and annexin V-positive cells of 4 independent experiments are shown in the graphs. ∗P < 0.05 vs. medium alone. (B) Western blot showing phosphorylation on Stat-3 in whole-cell lysate, Erk 1/2, and Akt after treatment with CT-1 at the indicated time. Subsequent stripping of blots followed by incubation with antibodies against corresponding specific molecules (lower respective panels) was performed to monitor protein load. The blots are representative of 4 experiments with similar results.
Gastroenterology  , DOI: ( /S (03)00698-X)

3. Figure 2
Effects of CT-1 on cultured rat hepatocytes. (A ) Western blot analysis showing phosphorylation of Stat-3, Erk 1/2, and Akt after stimulation with CT-1 (50 ng/mL) of primary hepatocytes maintained in serum-free culture for 48 hours. The figure is representative of 3 similar experiments. (B) Primary hepatocytes cultured for 12 hours in the absence or in the presence of CT-1 (at 2 different concentrations, 50 and 100 ng/mL) were challenged with TGF-β1 (5 ng/mL) for 12 additional hours and apoptosis was estimated by determining caspase-3 activation. Control indicates unchallenged cells cultured with medium alone. The value of caspase-3 activity of hepatocytes treated with TGF-β1 alone (TGF-β in the graph) was normalized to 100%. Data from 5 independent experiments are presented as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01 vs. TGF-β.
Gastroenterology  , DOI: ( /S (03)00698-X)

4. Figure 3
CT-1 is a hepatocyte survival-promoting factor up-regulated in cultured hepatocytes and in regenerating liver. (A ) RT-PCR for CT-1 in primary hepatocytes cultured in serum-free conditions. (B) Levels of CT-1 as estimated by ELISA in the supernatant of primary hepatocytes maintained in serum-free culture for 24 hours in the absence (medium) or presence of TGF-β (5 ng/mL) added to the culture 6 hours before cell harvesting. Data are the mean values ± SEM of 4 independent experiments with triplicates in each assay. ∗P < (C ) MTT assay of primary hepatocytes cultured in serum-free conditions for 24 hours showing the influence on cell survival of anti-CT-1 antibody (4 and 8 μg/mL) added to the medium. Data are means ± SEM of 7 independent experiments performed in triplicate. ∗P < 0.01 vs. control. (D) Northern blot analysis of CT-1 gene expression in rat liver (C ) and at different time points after partial hepatectomy (representative blot of 4 animals per time point).
Gastroenterology  , DOI: ( /S (03)00698-X)

5. Figure 4
Effect of AdCT-1 therapy in rats with fulminant hepatic failure after 90% hepatectomy. (A ) Survival of rats at 48 hours after 90% hepatectomy. No more deaths occurred beyond this time point. Two days before the surgery, rats received through the tail vein (•) AdCT-1 (n = 13), (▵) control vector AdLacZ (n = 14), or (○) saline (n = 10). P < 0.01 AdCT-1 group vs. AdLacZ or saline groups. (B) Western blot analysis of homogenates obtained from normal rat liver and from liver tissue sampled 1 hour after 90% hepatectomy in rats receiving AdCT-1, AdLacZ, or saline (n = 5 per group) 2 days before the surgery. Antibodies against total and phosphorylated Stat-3, Erk 1/2, and Akt were used. The blots are representative of 5 repeated experiments with consistent results. (C ) Caspase-3 activity in homogenates of liver tissue sampled 1 hour after 90% hepatectomy in rats that received AdCT-1 or control adenovirus AdLacZ or vehicle 2 days before the surgery (n = 5 per group) ∗P < 0.05 vs. saline.
Gastroenterology  , DOI: ( /S (03)00698-X)

6. Figure 5
Protective effect of AdCT-1 against acute liver injury induced in mice by administration of Con-A. (A ) Serum ALT level 6 hours after Con-A challenge in mice pretreated with AdCT-1, AdLacZ, or saline. ∗∗P < 0.01 vs. saline or AdLacZ. (B) TUNEL staining in liver sections 6 hours after challenge with Con-A in mice pretreated with AdCT-1, AdLacZ, or saline. (C ) Representative Western blotting of liver extracts at 1 hour after challenge with Con-A in animals pretreated with AdCT-1, AdLacZ, or saline (n = 5 per group). Antibodies against total and phosphorylated Erk 1/2 and Akt were used.
Gastroenterology  , DOI: ( /S (03)00698-X)

7. Figure 6
Protective effect of recombinant CT-1 against acute liver injury induced in mice by administration of Con-A. (A ) Serum ALT level 6 hours after Con-A challenge in mice pretreated with 5 μg of intravenous CT-1 or saline 20 minutes before Con-A challenge (n = 4 per group). ∗P < (B) TUNEL staining in liver sections 6 hours after challenge with Con-A in mice pretreated with CT-1 or saline.
Gastroenterology  , DOI: ( /S (03)00698-X)