Figure 1.
Expression of ductal markers in adult rat liver and pancreas tissue.
Immunohistochemistry for cytokeratin 7 (CK7), cytokeratin 19 (CK19), cytokeratin 20 (CK20), OV6 and Peanut Agglutinin (PNA) in adult rat liver and pancreas sections. A control (no primary antibody) is also shown. All scale bars, 100 µm.
Figure 2.
Differentiation of B13 cells to ductal and hepatic phenotypes.
Immunostaining for Amylase (red)/CK20 (green), TFN (red)/CK20 (green), PNA, OV6, CK7, Cx43 (green) in untreated (control, CTL), DEX, Dex/EGF and EGF treated B13 cells. Scale bars, first and second row, 20 µm; all others 40 µm.
Figure 3.
EGF enhances the ductal phenotype at the expense of the hepatic phenotype.
Bar charts showing the percentage of cells expressing (A) Amylase and Transferrin (B) CK7, CK20, PNA and OV-6 in control, EGF, Dex/EGF and Dex treated cells. (C) Scatter plot from the FACSCanto showing the intensity of PNA staining in Dex/EGF treated cells and bar charts showing percentage of cells positive for CK7 and Sox9 (positive fraction) following MACS isolation. Scale bars, top and middle row, 20 µm; lower row, 50 µm.
Figure 4.
Expression of ductal markers and inhibition of the ductal phenotype.
(A) RT-PCR for Cx43 and GSTπ (B) Western blotting analysis for Albumin, TFN, AFP and the liver enriched transcription factor C/EBPβ in control, EGF, Dex/EGF and Dex treated cells. β-actin and α-tubulin are also shown as loading controls. (C) Immunostaining for CK7 in control and Dex/EGF treated cells in the presence and absence and absence of the EGF receptor inhibitor AG1478. The inhibitor was added at a final concentration of 25 µM.
Figure 5.
Electron microscopy and stability of the ductal phenotype.
(A) Electron micrographs of control, Dex and Dex/EGF treated B13 cells. (B) Immunostaining for amylase, CK20 and CK7 following withdrawal of Dex and EGF in treated B13 cells. Control B13 cells are also shown (B). Scale bars for electron micrographs are (from left to right); 2, 2, 1 and 0.5 µm. Scale bars in second row, 20 µm and 40 µm for all others.
Figure 6.
Lineage trace of ductal phenotype.
(A) Infection of HepG2, control B13 and Dex/EGF treated B13 cells with Ad-CK19-nucGFP and (B) immunostaining for amylase and TFN in Dex/EGF treated B13 cells infected with Ad-CK19-nucGFP.
Figure 7.
CEBPβ controls the switch in phenotype from pancreatic B13 cells to hepatocyte or ductal cells.
Immunostaining for C/EBPβ/TFN and C/EBPβ/PNA in control, Dex and Dex/EGF treated cells (A) and after infection with Ad-CMV-LAP (B) and Ad-CMV-LIP (C). In A only the induced endogenous C/EBPβ is visible. Scale bars, 20µ m.
Figure 8.
Schematic representation of the possible pathways of differentiation of B13 cells into hepatocytes and ductal cells.
Diagram illustrating the potential relationship between pancreatic acinar cells, ductal cells and hepatocytes.