2 D,E). Melatonin levels were higher in supernatant of cholangiocytes from BDL, compared to healthy, rats and increased in cholangiocyte samples from BDL rats treated with melatonin (Supporting Table 1). Consistent with previous studies,16 melatonin serum levels were higher in BDL, compared to healthy, rats (Table 1). Melatonin serum levels increased GDC-0068 mouse in healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to rats treated with mismatch Morpholino (Table 1). Although AANAT biliary expression decreased in rats treated with AANAT Vivo-Morpholino (Fig. 2 A-C), the increase in melatonin serum levels observed in these rats was likely

a result of enhanced expression of AANAT (and subsequent increased melatonin secretion) in the pineal

gland and small intestine, which also express AANAT.13, 27 Melatonin levels decreased in supernatant of cholangiocytes from healthy and BDL rats treated Olaparib manufacturer with AANAT Vivo-Morpholino, compared to controls (Table 1). In liver sections from healthy and BDL rats treated with AANAT Vivo-Morpholino, there was increased percentage of PCNA-positive cholangiocytes and IBDM, compared to controls (Fig. 3A,B; Table 1). No changes in biliary apoptosis (Table 1) were observed between healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to healthy rats treated with mismatch Morpholino. No difference in lobular damage or necrosis was observed for healthy versus BDL rats treated with AANAT Vivo-Morpholino,

compared to controls (not shown). A similar degree of portal inflammation was observed between healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (not shown). Serum levels of transaminases, ALP, and TBIL decreased in BDL rats treated with Vivo-Morpholino, compared to rats treated with mismatch-Morpholino (Table 1). In BDL Mismatch-treated rats, we found that connective tissue represents approximately 1.5% of the liver, whereas in BDL rats treated with AANAT Vivo-Morpholino, collagen tissues represented approximately 3% of liver mass (not shown). None of the organs analyzed by H&E staining showed structural damage, necrosis, or inflammation (not shown). There was increased expression of mRNA (Fig. 4A) and protein (Fig. 4B) of PCNA, SR, CFTR, and Cl−/HCO AE2 in cholangiocytes MRIP from rats treated with AANAT Vivo-Morpholino, compared to controls (Fig. 4B). In vitro, melatonin inhibited biliary proliferation (by MTS assays and PCNA immunoblottings; Supporting Fig. 1) and protein expression (by FACS analysis) of SR, CFTR, and Cl−/HCO AE2, compared to large cholangiocytes treated with 0.2% BSA (Supporting Fig. 1). Enhanced mRNA and protein expression for AANAT and increased melatonin secretion were observed in AANAT-transfected cholangiocytes, compared to controls (Supporting Fig. 2A-C). In cholangiocytes overexpressing AANAT, there was (1) decreased biliary proliferation shown by PCNA immunoblottings and MTS assays (Fig.