1. A Mechanism of Cell Survival
Xue Wang, Marie C DeFrances, Yu Dai, Peter Pediaditakis, Carla Johnson, Aaron Bell, George K Michalopoulos, Reza Zarnegar 
Molecular Cell 
Volume 9, Issue 2, Pages (February 2002)
DOI: /S (02)

2. Figure 1
High HGF Levels Sensitize Hepatic Cells to Fas-Mediated Apoptosis by Promoting DISC Formation and by Activating the Initiator Caspase, Caspase-3
(A) Hepa1-6 cells were cultured in duplicate under serum-free conditions in the presence or absence of 50 ng/ml recombinant human dHGF, challenged with or without the addition of 500 ng/ml Jo2 anti-Fas monoclonal antibody, and incubated for the indicated time. The extent of apoptosis was determined by Trypan blue dye exclusion, and the data represent the average of three different independent experiments (mean ± SEM). The values for dHGF treatment (+dHGF) are significantly different (*p < 0.05) from the control without dHGF addition (−dHGF) using the unpaired Student's t test. Hepa1-6 cells were serum-starved overnight and then cultured in the presence of dHGF for various lengths of time as indicated. Their lysates were then subjected to immunoprecipitation and Western blot analysis as indicated to detect caspase-8, FADD, Fas (B), and caspase-3 activation (C).
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2
Met and Fas Preexist as a Complex and Are Physically Associated with Each Other
(A) Hepa1-6 cells were incubated with dHGF (50 ng/ml) or Jo2 antibody (200 ng/ml) for various lengths of time, and the cell lysates were subjected to immunoprecipitation and Western blot analysis for Met and Fas as indicated in the figure.
(B) Schematic diagram of the structural domains of HGF and its naturally occurring isoforms, NK1 and NK2 (K denotes kringle domains, and the triangle represents the hair pin loop in the amino-terminal portion of the α chain).
(C) Hepa1-6 cells were treated with the indicated ligand (dHGF, 50 ng/ml; NK1 and NK2, 100 ng/ml; or FasL, 200 ng/ml) for 15 min. Their lysates were subjected to immunoprecipitation using anti-Fas or anti-phosphotyrosine antibodies and Western immunoblot using anti-Met antibody as indicated.
(D) Equal aliquots of lysate from Hepa1-6 cells were subjected to immunoprecepitation (IP) experiments and Western blot analysis using the indicated antibodies. “p” and “s” denote pellet and the supernatant recovered after IP, respectively.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3 Met Binding to Fas Inhibits FasL Binding to Fas
Fas ligand binding studies were carried out on Hepa1-6 cells in culture as described under Experimental Procedures. The data represent the average of two independent experiments that had similar results. An asterisk indicates the presence of a statistically significant difference (p < 0.05 Student's t test) in FasL binding in the dHGF pretreated (15 min, 50 ng/ml) group versus the untreated control group.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 The Extracellular Domain of Met Directly Binds to Fas
(A) HepG2 cells in culture were treated with the crosslinker DSS (Disuccinimidyl suberate). Cell lysate was subsequently prepared and subjected to immunoprecipitation and SDS-PAGE under nonreducing conditions. After sample transfer to PVDF membrane, the blot was cut into half and probed with the indicated antibody. The Met-Fas crosslinked complex (Mr 230 kDa) is shown with an arrowhead. NS denotes a nonspecific band migrating at the bottom of the gel and is shown for equal loading.
(B) In vitro-translated 35S-labeled Ex-Met was incubated with purified recombinant Fas-Fc chimeric fusion protein, subjected to pull-down experiments using protein-A agarose, and analyzed by SDS-PAGE and autoradiography as described under Experimental Procedures. Positive (anti-Ex-Met antibody) and negative (normal rabbit IgG) controls were used in parallel as indicated in the figure. The arrow indicates the 110 kDa Ex-Met protein.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5
Cells Expressing the Extracellular Domain of Met (Ex-Met) Are Resistant to Apoptosis Induced by Fas Agonist and Are Refractory to Met-Fas Dissociation Elicited by dHGF or Jo2 Antibody
(A) Hepa1-6 cells (clone ) stably transfected with the tet-on expression vector encoding the extracellular portion of Met (Ex-Met) were cultured in the presence of doxycycline to maximally induce Ex-Met. Cell lysate was prepared and subjected to immunoprecipitation with anti-Fas or normal rabbit IgG and analyzed by Western blot using affinity-purified anti-Met antibody raised against the C-terminal tail of truncated Met (Ex-Met). Lysate from the parental Hepa1-6 cells was also included as a control. The p110 band corresponding to extracellular Met is shown.
(B) Cells were subjected to the cell-impermeable thio-cleavable crosslinking agent DSSTP in culture. After crosslinking, cell lysate was subjected to SDS-PAGE under reducing or nonreducing conditions as indicated. The resulting blot was cut into half (samples were loaded in duplicate) and probed with either anti-Ex-Met or anti-Fas as depicted. The Met-Fas and the Ex-Met-Fas complexes are indicated by an arrowhead. NS denotes a nonspecific band migrating at 50 kDa (most likely IgG) and is shown for equal loading.
(C) Cells were cultured in duplicate in the presence or absence of doxycycline (1 μg/ml) to maximally induce Ex-Met and were then treated with 500 ng/ml Jo2 monoclonal anti-Fas agonistic antibody for the indicated time. The extent of apoptosis was determined. Error bars represent mean percent apoptosis ± SEM. The values for the two groups were significantly (*p < 0.05) different as determined by the unpaired Student's t test.
(D) Ex-Met expressing Hepa1-6 cells were treated with either dHGF (50 ng/ml) or Jo2 antibody (200 ng/ml) for various lengths of time, and their lysates were subjected to antibody pull-down experiments using anti-Fas antibody and Western blot analysis using commercially available antibody against the C-terminal end of full-length Met.
(E) Cells were incubated with 125I-dHGF (50 ng/ml) at 4°C for 1 hr and then subjected to crosslinking with DSS; cell lysates were subsequently prepared and subjected to immunoprecipitation, SDS-PAGE, and autoradiography.
(F) The Hepa1-6 cells expressing Ex-Met were cultured in the presence or absence of doxycycline (1 μg/ml) for 48 hr to maximally induce Ex-Met and were treated with (50 ng/ml) or without dHGF for the indicated lengths of time. Their lysates were subjected to immunoprecipitation with anti-phosphotyrosine and Western immunoblotting with anti-Met as indicated.
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6
Transgenic Mice Expressing the Extracellular Portion of Met (Ex-Met) in the Liver Are Resistant to Fas-Induced Hepatocyte Apoptosis
(A) Transgenic mice expressing Ex-Met under the transcriptional regulation of the albumin promoter and enhancer (Ex-Met-TG) (n = 5) and their nontransgenic littermates (NTG) (n = 9) were injected intravenously with Jo2 antibody (0.2 μg/g body weight in 60 μl of saline). They were closely monitored for overall survival (A) and analyzed for liver damage and hepatocyte apoptosis using immunohistologic (B) and biochemical methods (caspase-3 activity) (C). The transgenic mice were significantly resistant to Fas-induced death (p < 0.02 for survival rate as determined by the Fisher's exact test). The transgenic mouse livers contained significantly (p < 0.02) fewer apoptotic hepatocytes and reduced caspase activity (p < 0.05), respectively, as compared to the nontransgenic littermates using the unpaired Student's t test. Apoptotic nuclei (shown by arrows) were identified by TUNEL staining, and the data are presented as mean percent apoptosis following evaluation of at least 2,000 hepatocytes/section in randomly selected fields from the livers of transgenic and nontransgenic animals. Caspase-3 activity was determined using the caspase assay kit from PharMingen. Enzyme activity is presented as mean relative fluorescence from transgenic and nontransgenic animals (n = 5).
Molecular Cell 2002 9, DOI: ( /S (02) )

8. Figure 7 A Model for Met-Induced Fas Sequestration and Cell Survival
For an explanation, please see text.
Molecular Cell 2002 9, DOI: ( /S (02) )