Volume 129, Issue 5, Pages 1633-1642 (November 2005) Expression and Role of Gas6 Protein and of Its Receptor Axl in Hepatic Regeneration From Oval Cells in the Rat  Dominique Couchie, Fouad Lafdil, Nadine Martin–Garcia, Yannick Laperche, Elie Serge Zafrani, Philippe Mavier  Gastroenterology  Volume 129, Issue 5, Pages 1633-1642 (November 2005) DOI: 10.1053/j.gastro.2005.08.004 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Gas6 and Axl mRNA expression in hepatic regeneration from oval cells. Gas6 (hatched bar) and Axl (open bar) mRNA expression was measured as described in the Materials and Methods section. Results are expressed as fold increase over basal value obtained in normal rat livers (304 ± 45 and 21 ± 3 mRNA copies per ng of total RNA for Gas6 and Axl, respectively; ten experiments). Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Gas6 mRNA detection by in situ hybridization and immunohistochemical expression of Gas6 and cytokeratin 19 in hepatic regeneration from oval cells. (A) Gas6 mRNA is detected with an antisense riboprobe in periportal oval cells forming ductular structures (day 5 after partial hepatectomy, original magnification, ×400). (B) Gas6 mRNA is detected with an antisense riboprobe in oval cells forming ductular structures as well as in spindle-shaped cells surrounding them (day 9 after partial hepatectomy, original magnification, ×1000). (C) Absence of Gas6 mRNA expression with a sense riboprobe (day 5 after partial hepatectomy, original magnification, ×400). (D–F): expression of cytokeratin 19 (D, green fluorescence) and Gas6 (E, red fluorescence) and their colocalization (F) in ductular structures (day 9 after partial hepatectomy, frozen material, original magnification, ×400). Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Immunohistochemical detection of Axl and cytokeratin 19 in hepatic regeneration from oval cells at day 9 after partial hepatectomy. (A) Axl expression, detected on paraffin-embedded material by an immunoperoxidase method, predominates in periportal regions (original magnification, ×100). (B) Axl is mainly expressed in oval cells forming periportal ductular structures (original magnification, ×400). (C–E) Expression of cytokeratin 19 (C, green fluorescence) and Axl (D, red fluorescence) and their colocalization (E) (frozen material, original magnification, ×200). Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Expression of Gas6 and Axl proteins in rat WB-F344 cells. Western blot analysis of Gas6 (A) and Axl (B) proteins was performed as described in the Materials and Methods section. In lanes 2 and 4, the antibody was preincubated with an excess of a specific blocking peptide. Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of Gas6 on the number and viability of rat WB-F344 cells. Cells were cultured for 6 days in the absence (○, ●) or in the presence (□, ■) of 0.1 % fetal calf serum and incubated with (●, ■) or without (○, □) Gas6 (400 ng/mL). Cell viability was determined at days 0, 2, 4, and 6 as described in the Materials and Methods section (10 experiments). *P < .01 when compared with 0% fetal calf serum without Gas6. #P < .05 when compared with 0.1% fetal calf serum without Gas6. NS, not significant. Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Effect of Gas6 on [3H]-thymidine incorporation by WB-F344 cells. Serum-starved confluent cells were cultured with or without fetal calf serum in the absence (hatched bar) or in the presence (open bar) of Gas6 (400 ng/mL). [3H]-thymidine incorporation was determined as described in the Materials and Methods section (6 experiments). Results are expressed as the percentage of the incorporation obtained in cells stimulated with 5% fetal calf serum (119,455 ± 25,549 cpm). #P < .05 when compared with 0% fetal calf serum without Gas6. NS, not significant. Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effect of Gas6 on WB-F344 cell apoptosis. (A) DAPI staining of nuclei (original magnification, ×400). Serum-starved cells were treated for 17 hours with (b,c) or without (a) 5 μmol/L 15-d-PGJ2 in the absence (b) or in the presence (c) of Gas6 (400 ng/mL). (B) Caspase-3-like activity. Serum-starved cells were treated for 8 hours with 5 μmol/L 15-d-PGJ2 in the absence or in the presence of Gas6 (400 ng/mL). Caspase-3-like activity was assayed as described in the Materials and Methods section and expressed as fluorescence arbitrary units/mg of protein/minute (12 experiments). *P < .01 when compared with 15-d-PGJ2 alone. Gastroenterology 2005 129, 1633-1642DOI: (10.1053/j.gastro.2005.08.004) Copyright © 2005 American Gastroenterological Association Terms and Conditions