Inverse correlation between the concentration of HGF in culture and production of HGF by HSCs.

<p>(<b>A</b>) Real-time PCR for HGF mRNA shows a dose-dependent decrease in HGF production by HSC-T6 cells in response to increasing concentrations of HGF in culture (*P<0.05; **P<0.01). (<b>B</b>) HGF protein production by HSC-T6 cells decreases in response to increased HGF, as measured by WB. Actin represents loading control. (<b>C</b>) Densitometry analysis on representative WB shown in (B).</p

Recombination occurs in all hepatic cell populations, including those that produce HGF.

<p>(<b>A</b>) Separation of hepatic cell populations from HGF^{ex.5 flox};Mx1-cre mice into hepatocytes and NPCs shows recombination in both after p(I):p(C) treatment as compared to controls. (<b>B</b>) RT-PCR shows persistence of unrecombined HGF mRNA in both hepatocytes and NPCs. (<b>C</b>) Real-time PCR for full-length HGF mRNA shows a decrease in HGF in the NPC fraction after p(I):p(C) treatment. (<b>D</b>) WB for HGF in hepatocytes and NPCs shows that the amount of HGF is unchanged in KOs compared to controls, and is found mainly in the NPC fraction. Ponceau represents loading control.</p

Genotype and number of mice used in experiments.

<p>Genotype and number of mice used in experiments.</p

Liver regeneration stimulated by CCl4 depletes HGF mRNA and protein in HGF KO mice.

<p>(<b>A</b>) CCl4 treatment following genomic recombination further decreases full-length HGF mRNA, as assessed by real-time PCR. (<b>B</b>) WB shows decreased HGF expression in livers of HGF KO mice treated with CCl4 in combination with p(I):p(C), as compared to controls or those treated with p(I):p(C) only. Ponceau represents loading control.</p

Persistence of unrecombined HGF mRNA and protein in the livers of HGF^{ex.5 flox}; Cre^+/− mice after genomic recombination.

<p>(<b>A</b>) Schematic of the targeting strategy for conditional inactivation of the gene for HGF (top). Cre-mediated excision of the floxed HGF allele (middle) leads to the generation of a recombined allele (bottom) lacking exon 5. (<b>B</b>) Successful genomic deletion of HGF exon 5 after induction of recombination, as shown by PCR. Top - HGF^{ex.5 flox};Cre-ER^T mice; bottom - HGF^{ex.5 flox};Mx1-cre mice. (<b>C</b>) RT-PCR shows the presence of both recombined and unrecombined HGF mRNA in KO livers. (<b>D</b>) WB for HGF in control and HGF KO livers shows no differences after recombination. Ponceau represents loading control.</p

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

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