Figure 2.

Cholangiocytes Upregulate FN in Response to TGFβ.

A. H69 cells treated for 24 hours with several agonists prominent in biliary disease show that only TGFβ stimulates FN production. TGFβ: 10ng/ml, TLCA: 20uM, LPS: 200ng/ml, IL-6: 50ng/ml. Western blot for FN and EZH2 with GAPDH as a loading control. These expression levels were quantified by densitometry (right). *p<0.01. All error bars are SEM, n=3.

B. Western blot showing repression of TGFβ–stimulated FN by overexpression of EZH2 using adenoviral constructs (AdEZH2) (left). Quantification of the blot for EZH2 and FN normalized to LacZ-vehicle. *p<0.01 and **p<0.001. All error bars are SEM, n=4.

C. TGFβ treatment for 24 hours stimulates FN production in cholangiocytes and a reduction in total EZH2 levels. Immunoblotting for FN and EZH2 on H69 cells treated with TGFβ for 24 hours (left). These were quantified by densitometry for FN and EZH2 (right) *p<0.01 compared to vehicle. All error bars are SEM, n=3.

D. RT-PCR analysis on H69 cells treated with TGFβ for 24 hours shows an increase in FN gene expression with no change in EZH2 gene expression. *p<0.01 compared to vehicle. All error bars are SEM, n=3.

E. Cholangiocytes isolated from WT miee were treated with 10ng/ml TGFβ for 24 hours. Western blotting for FN and EZH2 show TGFβ -stimulated FN production with a concurrent decrease in EZH2 protein (left). Densitometry from 3 different experiments shows an increase in FN and a decrease in EZH2 protein (right). *p<0.01 compared to vehicle. All error bars are SEM, n=3.

F. RT-PCR analysis on mouse cholangiocytes treated with TGFβ shows an approximately 2-fold increase in FN gene transcription with no change in EZH2 gene transcription. *p<0.01 compared to vehicle. All error bars are SEM, n=3.