2A). In both models Q-PCR (Fig. 1C and Supporting Fig. 2A, right panels) showed that the mRNA levels of both HAIs were significantly up-regulated in the livers of mice receiving bile-duct ligation or rotavirus infection, a phenomenon similar to observations in human BA. Using immunohistochemistry (IHC), we also evaluated the expression of both HAIs in the livers of other human cholangiopathies, including primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and intrahepatic cholestasis in children, such as progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis

(BRIC). An increased number of ductular cells positive for CK19, HAI-1, and HAI-2 (Fig. 2A for PBC and PSC) was found in the livers of cholangiopathies except BRIC, apparently with different incidence (Fig. 2B). Because cryopreserved ABT-737 mouse human tissues were not available, we instead employed a xenobiotic-induced PBC mouse model to assay HAI expression23 and found that the expression of both HAIs was also elevated in PBC mice (Supporting Fig. 2B). In contrast, we could not detect any increase in HAI expression in the only two cryopreserved liver biopsies of type-II PFIC available (Supporting Fig.

2C) compared with those in a near-normal liver and an NH liver. This was consistent with the IHC result that showed no increase of CK19-positive ductular cells in type-II PFIC (Supporting Fig. 2D). In a type-III PFIC CX-5461 liver (Supporting Fig. 2E,F), however, the ductular reactions were seen with increased expression of CK19, HAI-1, and HAI-2. To further identify which types of cells expressed abundant HAI-1 and -2 in BA livers, we performed colocalization studies and showed that both HAI-1 (Fig. 2C, left) and HAI-2 Phospholipase D1 (Fig. 2C, middle) were coexpressed with CK19, a well-known marker for HSCs and cells of the cholangiocyte lineage.24 Moreover, HAI-1 was also coexpressed with epithelial cell adhesion molecule (EpCAM), Gli-2, and OV6, additional biomarkers for

HSCs (Supporting Fig. 3A-C). Because the majority (>90%) of cells expressing HAI-1 also coexpressed HAI-2 in BA livers (Fig. 2C, right), we assumed that HAI-2 was also coexpressed in most EpCAM-, Gli-2-, and OV6-expressing cells, although colocalization studies could not be performed as these antibodies were raised in the same animal species. In addition, HAI-1 was occasionally found in the cells expressing α-fetoprotein (AFP), a marker for hepatoblasts (Supporting Fig. 3D). Therefore, HAI-1 and -2 were expressed mostly in cells of cholangiocyte lineage and HSCs. Because several proinflammatory cytokines,25 growth factors,26 and bile acids27 have been found elevated in the serum and/or liver of BA patients, we next determined whether these factors are involved in activating HAI expression in BA livers.