1. Volume 125, Issue 5, Pages 1303-1310 (November 2003)
Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat1  
Tatyana V Masyuk, Bing Q Huang, Christopher J Ward, Anatoliy I Masyuk, David Yuan, Patrick L Splinter, Rachaneekorn Punyashthiti, Eric L Ritman, Vicente E Torres, Peter C Harris, Nicholas F Larusso 
Gastroenterology 
Volume 125, Issue 5, Pages (November 2003)
DOI: /j.gastro

2. Figure 1
Liver pathology in PCK rats. (A) Intrahepatic biliary trees of normal and (B) PCK rats were scanned using a microcomputed tomography technique that allows reconstruction in 3 dimensions of the bile duct network under conditions that preserve its original morphology (scale bars, 1 mm). Architecture of the PCK intrahepatic biliary tree is changed substantially owing to bile duct dilatation and increased biliary tree volume. Inset shows high-magnification image. (C) Part of biliary tree isolated from PCK rat by microdissection provides anatomic evidence of pathologic changes in biliary tree morphology with a cluster of cysts (arrow) and normal bile duct (asterisk) shown. (D) SEM of liver from normal and (E) PCK rats (magnification 200×; pv, portal vein; ha, hepatic artery; bd, bile duct; Cy, cyst). (F) The biliary origin of the cystic epithelium was confirmed by staining with CK-19, a marker of bile duct epithelial cells. (A) Reprinted with permission.22
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Cilia in normal cholangiocytes. (A) SEM and (B) TEM show that cholangiocytes of IBDUs express primary cilia on the apical surface. Cross-section confirmed the ultrastructure of primary cilia (B, inset). Scale bars, 1 μm.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Western blot analysis of fibrocystin expression in (A) human and (B) rat liver and kidney with the monoclonal antibody FB2. (A) Two bands at greater than 450 kilodaltons and approximately 200 kilodaltons were seen in both normal human kidney and liver. In the kidney, the major band was at greater than 450 kilodaltons, but in the liver the major protein was approximately 200 kilodaltons. The loss of fibrocystin expression in ARPKD liver provides evidence of antibody specificity. (B) In the rat, the major proteins in normal kidney (>450 kilodaltons) and liver (∼200 kilodaltons) parallel those seen in humans. In the PCK rat continued expression of fibrocystin was present in the kidney and liver, suggesting that not all Pkhd1 spliced forms are lost in the PCK rat (which is homozygous for a splicing mutation in intron 35).
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Fibrocystin is localized to primary cilia in normal cholangiocytes of IBDUs. (A) Immunofluorescent images of freshly isolated intrahepatic bile ducts from normal rat labeled with monoclonal antibodies to acetylated α-tubulin (red) and fibrocystin (green). Their colocalization to cholangiocyte primary cilia is shown as yellow with 4′,6-diamidino-2-phenylindole staining of the nuclei. Images were captured at the level of the apical membrane. (B) Expression of fibrocystin in primary cilia by IEM using FB2. Scale bar, 1 μm.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Cilia in the PCK rat are shortened, malformed, and do not express fibrocystin. (A, B) By SEM, primary cilia on the apical surface of cystic epithelia from the PCK rat were found to be more variable in length than in normal cholangiocytes. (B) Inset is a short cilium at high magnification. (C, D) TEM images show ciliary abnormalities such as a bulbous extension of the axonemal membrane (A and D, inset). (E) Immunostaining for acetylated α-tubulin, (F) fibrocystin, and (G) colocalization in cystic epithelia shows that fibrocystin is undetectable in cilia. (H) Presence of fibrocystin in the cytoplasm of cholangiocytes in the PCK rat. Scale bars, 1 μm.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Inactivation of Pkhd1 by siRNA results in a decrease of messenger RNA level and ciliary length, and loss of fibrocystin expression. (A) An siRNA targeting Pkhd1, but not a scrambled siRNA, decreased the messenger RNA level by 80%. (B) SEM showed that primary cilia (asterisks) in IBDUs treated with siRNA are decreased in length by an average of 41% compared with cilia in IBDUs treated with scrambled siRNA (C). (D–F) Only acetylated α-tubulin (D) but not fibrocystin (E) was found in cholangiocyte cilia treated with siRNA against Pkhd1. (F) Merged images with 4′,6-diamidino-2-phenylindole staining of the nuclei. Scale bars, 1 μm.
Gastroenterology  , DOI: ( /j.gastro )