1. Liver Fibrosis Protects Mice From Acute Hepatocellular Injury
Éric Bourbonnais, Valérie–Ann Raymond, Chantal Éthier, Bich N. Nguyen, Marc Saba El–Leil, Sylvain Meloche, Marc Bilodeau 
Gastroenterology 
Volume 142, Issue 1, Pages e4 (January 2012)
DOI: /j.gastro
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2. Figure 1
Protective effect of COL1 on hepatocyte cultures. (A) Dose-response curve of COL1 (0.003 to 139 μg/cm2) on Balb/c mouse hepatocytes exposed to a single dose of Fas (250 ng/mL) for 24 hours. Hepatocytes plated on plastic dishes were used as control. (B) Time-response curve of a fixed dose (13.9 μg/cm2) of COL1 following Fas stimulation (250 ng/mL). (C) Catalytic activities of activator capase-3 and effector caspase-8 obtained from hepatocytes plated on plastic or COL1 following Fas stimulation. (D) Apoptotic response of hepatocytes exposed or not to COL1 following Fas stimulation for 8 hours or exposed to actinomycin D (10 nmol/L) for 30 minutes followed by actinomycin D (10 nmol/L) plus tumor necrosis factor α (25 ng/mL) for 20 hours. Effect of (E) COL1 on viability and (F) ALT release of hepatocytes exposed to tert-butyl hydroperoxide for 1 hour. Results are expressed as mean ± SEM from at least 4 different experiments. *P < .05, **P < .01, ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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3. Figure 2
Expression of proapoptotic and antiapoptotic proteins on COL1 exposure. Western blots performed on protein extracts of cultures of Balb/c mouse hepatocytes plated on plastic or COL1 (13.9 μg/cm2) for 4 hours after attachment. (A) Bcl-xL, (B) Bak, (C) Bad, (D) Bax, and (E) Bid. β-actin was used as control. Results are expressed as mean ± SEM from at least 4 different experiments. *P < .05, ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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4. Figure 3
ERK expression in hepatocytes exposed to COL1. Balb/c mouse hepatocytes were plated on plastic or COL1-coated dishes. After attachment in Williams' E medium containing 10% foetal bovine serum, medium was changed and replaced with serum-free fresh medium overnight. (A) U0126 (25 μmol/L) was used to inhibit MAPK for 1 hour before the next change for fresh serum-free media and beginning of the kinetic analysis for 4 hours. (B) U0126 or PD98059 were incubated for 1 hour before the beginning of the experiment for 4 hours. Blots represent the levels of pERK and ERK activities in protein extracts from hepatocyte cultures. (C) Effect of MAPK pathway inhibition by U0126 for 1 hour on Bax and Bid protein expression from hepatocytes plated on COL1-coated dishes for 4 hours. Plastic was used as control. (D) Apoptotic index of hepatocytes treated with U0126 or PD98059 for 1 hour before Fas stimulation for 6 hours on plastic or COL1-coated dishes. (E) Effect of collagen on Fas-induced apoptosis on hepatocytes isolated from control CD1 (ERK1&2+/+) mice and on ERK1&2+/−, ERK1+/−, and ERK1−/− animals for 6 hours. Results are expressed as mean ± SEM from at least 4 different experiments. *P < .05, **P < .01, ***P < .001.
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5. Figure 4
Protective effect of fibrosis against Fas-induced injury in vivo. (A) Measure of hydroxyproline content in livers of animals following 1 month, 3 months, and 3 months with a 2-week recuperation period of TAA (200 μg/g body wt, 3 times a week) or saline treatments. (B) Phospho-ERK1 and 2 protein expression following TAA or saline treatments for 3 months or 3 months with a 2-week recuperation period treatment. Representative blots of phospho- and total ERK1 and 2. (C) ALT release after 3 months of TAA or saline treatment followed, 48 hours after the last TAA or saline injection, by Fas injection (0.5 μg/g body wt) for 6 hours. (D) Catalytic activity of caspase-3 in livers of mice treated with TAA or saline for 3 months followed by Fas injection for 6 hours. (E) ALT release after 8 weeks of CCl4 treatment or vehicle followed, 48 hours after the last CCl4 or mineral oil injection, by a single dose of Fas (0.5 μg/g body wt). Animals were killed 6 hours after Fas injection. Controls were obtained from non-TAA, non-Fas–treated animals. (F) ALT release from control CD1 (ERK1&2+/+) mice and ERK1−/− animals after 3 months of TAA or saline treatment followed, 48 hours after the last TAA or saline injection, by Fas injection (0.5 μg/g body wt) for 6 hours. Results are expressed as mean ± SEM from at least 8 animals. *P < .05, **P < .01, ***P < .001.
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6. Figure 5
Representative microphotographs of histologic sections of mice livers. (A and B) Masson-trichrome stain and (C–F) hematoxylin-phloxin-safranin stain. (A) Saline-treated animal. (B) Three-month TAA-treated animal. (C) CD1 saline-treated animal. (D) CD1 3-month TAA-treated animal. (E) ERK1−/− animal with saline treatment. (F) ERK1−/− animal with 3-month TAA treatment. All animals received a single dose of Fas and were killed 6 hours later. The arrows represent fibrotic tissue. Original magnification 400×.
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7. Supplementary Figure 1
Proliferative status of mouse hepatocytes. 3H-Thymidine (1 μCi/mL) incorporation was measured on Hepa 1-6 murine cell line and primary mouse Balb/c hepatocytes plated on plastic and on increasing doses of COL1 for 24 and 48 hours.
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8. Supplementary Figure 2
AKT expression in vitro and in vivo. (Top) Western blots of phospho-AKT and AKT performed on protein extracts of cultured Balb/c mouse hepatocytes plated on plastic or COL1 (13.9 μg/cm2) for 4 hours after attachment. (Bottom) pAKT and AKT protein expression following TAA or saline treatments for 3 months. Representation blots of phospho-AKT and total AKT in the same conditions. **P < .01.
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9. Supplementary Figure 3
Kinetic of TAA-induced fibrosis. (A) Measurement of hydroxyproline content in livers of Balb/c animals after 4, 8, 10, 12, 14, and 12 weeks followed by a 2-week recuperation period of TAA (200 μg/g body wt 3 times a week) or saline treatment. (B) Serum ALT levels were measured from TAA- or saline-treated animals 48 hours after the last TAA or saline injection, followed by a single dose of Fas (0.5 μg/g body wt) for 6 hours. (C) Serum AST levels were measured from TAA- or saline-treated animals 48 hours after the last TAA or saline injection, followed by a single dose of Fas (0.5 μg/g body wt) for 6 hours.
Gastroenterology  , e4DOI: ( /j.gastro )
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10. Supplementary Figure 4
Expression of proapoptotic and antiapoptotic proteins on saline or fibrosis treatments. Western blots performed on livers of mice treated with thrice-weekly doses of saline or TAA for 3 months. (A) Bcl-xL, (B) Bak, (C) Bad, (D) Bax, and (E) Bid. β-actin was used as control. Results are expressed as mean ± SEM from at least 8 different animals. *P < .05, **P < .01.
Gastroenterology  , e4DOI: ( /j.gastro )
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11. Supplementary Figure 5
Hydroxyproline content and serum AST enzymes in ERK−/− animals and their controls. (A) Measurement of hydroxyproline content in livers of CD1 control and ERK1−/− animals following 12 weeks of TAA (200 μg/g body wt 3 times a week) or saline treatment. (B) Serum AST levels were measured from TAA- or saline-treated animals 48 hours after the last TAA or saline injection, followed by a single dose of Fas (0.5 μg/g body wt) for 6 hours.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions