Journal: Journal of Cellular and Molecular Medicine

Article Title: Oval cell proliferation in p16 INK4a expressing mouse liver is triggered by chronic growth stimuli

doi: 10.1111/j.1582-4934.2007.00178.x

Figure Lengend Snippet: Antibodies used in western analysis (WB) and immunohistochemistry (IHC)

Article Snippet: Cyclin D1 , 1:1000 (WB) , Stressgen (Ann Arbor, MI, USA).

Techniques: Western Blot, Immunohistochemistry, Transduction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Oval cell proliferation in p16 INK4a expressing mouse liver is triggered by chronic growth stimuli

doi: 10.1111/j.1582-4934.2007.00178.x

Figure Lengend Snippet: Liver-specific stimulation of proliferation by nodularin.Mice were treated with nodularin thrice weekly and killed next day after the last injection (NDLN) or 4 weeks after the last injection (NDLN + ). ( A ) Cyclin D1 mRNA levels were determined by real time RT-PCR. Differences between untreated p16 INK4a expressing and control mice, between controls and NDLN-treated controls (NDLN), and between nodularin treated controls that were killed 4 weeks after NDLN treatment were statistically significant ( P < 0.05, Student's t-test). The number of different animals tested was: p16(untreated) = 7, control(untreated) = 5, p16(NDLN) = 8, control(NDLN) = 12, p16(NDLN + ) = 21, control (NDLN + ) = 12.( B ) in situ immunodetection of PCNA ( B1 , B2 ), A6 antigen ( B3 , B4 ) and E-Cadherin ( B5 , B6 ) in nodularin treated mice.80-90% of hepatocytes nuclei of control mice were immunostained with the PCNA antibody (brown colour, B2 ). In p16 INK4a expressing mice predominantly small cells with oval nuclei were positively stained with the PCNA antibody (white arrow) and some of the hypertrophic hepatocytes ( B1 , black arrows), but less intensive than hepatocytes in control mice ( B2 , black arrows). The anti-A6 antibody identifies these cells as facultative liver stem cells, oval cells, forming ductular structures ( B3 , white polygon). These bipotent facultative stem cells of liver express the A6 antigen like cells of the biliary system (cholangiocytes, B3 , B4 , black polygon). A common feature of epithelial cells, hepatocytes, cholangiocytes and oval cells, in liver is the expression of E-cadherin.On the histological level E-cadherin is detected in periportal hepatocytes ( B6 , black arrowhead) biliary ductuli ( B5 , B6 , black polygon) and oval cells ( B5 , white polygons), whereas the latter form the same ductular structures detected with the A6 antibody ( B3 ).( C ) Nodularin treated p16 INK4a expressing mice ( C1 , C2 ) develop p16 INK4a negative loci ( C2 ) consisting of accumulating hypertrophic hepa-tocytes. Some of the hypertrophic hepatocytes were PCNA positive ( C1 , brown). The black lines mark the two foci in the PCNA stained liver slide ( C1 ). ( D ) Oval cells were isolated from nodularin treated p16 INK4a expressing mice by two-step collagenase perfusion and subsequently density gradient centrifugation. A6-positive cells (red colour) of passage 0 ( D1 ) growing clonally were trypsinated and cultured to passage 10 ( D2 ) without loss of A6 antigen. In higher passages ( D3 , passage 16) a loss or down-regulation of A6 expression was observed.Representative photomicrographs of OVUE265 are displayed. Bar represents 50 μm.

Article Snippet: Cyclin D1 , 1:1000 (WB) , Stressgen (Ann Arbor, MI, USA).

Techniques: Injection, Quantitative RT-PCR, Expressing, Control, In Situ, Immunodetection, Staining, Isolation, Gradient Centrifugation, Cell Culture