Am J Pathol. and cell differentiation. INTRODUCTION Pluripotent progenitor cells are crucial elements in regenerative medicine. Many progenitor cells were developed for numerous tissues including the liver: oval cells [1C3], liver epithelial cells [4C9] and small hepatocyte-like cells [10]. Improvements in Amuvatinib hydrochloride liver progenitor cell research may lead to new cell therapies and facilitate the development of new drugs [11C13]. However, many of the liver progenitor cells were very hard to isolate due to limited liver progenitor cell markers. Thus, a proper liver progenitor cell marker is usually highly desired to accelerate the development of liver regenerative medicine. We have previously derived an adult hepatic progenitor cell collection Lig-8 from your non-parenchymal portion of liver cells prepared from Fischer 344 rats [14, 15]. The Lig-8 cells share many properties Amuvatinib hydrochloride of the well-known liver progenitor cells WB-F344 [4C7] including epitheloid morphology, growth, and expression of hepatocyte or cholangiocyte markers: alpha fetal protein (AFP), albumin, alpha 1-antitrypsin, H.4 antigen, cytokeratin 8, cytochrome P 450 and cytokeratin 7 [4, 16, 17]. These cells can differentiate bi-potentially into hepatocyte- or cholangiocyte-lineage cells following induction by sodium butyrate (SB), a histone deacetylase inhibitor known to impact gene expression, inhibit proliferation and induce differentiation [6, 17, 18]. To Rabbit Polyclonal to EKI2 identify potential liver progenitor cell markers, we required advantage of a monoclonal antibody Ligab previously generated in our lab using whole Lig-8 cells [17]. The Ligab antibody reacts with the liver progenitor cells Lig-8 but not mature hepatocytes, suggesting that this Lig-8 cells express certain Ligab antigens specific to liver progenitor cells. Moreover, the expression of the Ligab antigens in the Lig-8 cells decreased when the cells underwent SB-induced cell differentiation [17]. Thus, the Ligab antigens could be potential liver progenitor cell markers. Using proteomics, we recognized brain isoform glycogen phosphorylase (GPBB) in a protein complex of the Ligab immunoprecipitates from your Lig-8 cells. Immunoblotting showed that GPBB was expressed in the Lig-8 and WB-F344 cells and the levels of GPBB in these cells decreased upon SB-induced cell differentiation, consistent with GPBB as a liver progenitor cell marker. GP is the first enzyme required for glycogenolysis [19]. Our shRNA-mediated GPBB knockdown followed by functional assays shows that GPBB facilitates liver progenitor cell survival under low glucose conditions and SB-induced cell differentiation. MATERIALS AND METHODS Cell culture and induction of cell differentiation Lig-8 cells were derived and cultured as previously explained [16, 17]. Cells between 29 and 35 passages were used. WB-F344 cells (courtesy of William B. Coleman, University or college of North Carolina at Chapel Hill, Chapel Hill, NC, USA) [5, 7, 20] were cultured in Dulbeccos Modified Eagle Medium (DMEM)/F12 made up of 10% fetal bovine serum (FBS), 20 mM HEPES (USB Corporation, Cleveland, OH, USA), and 1 penicillin-streptomycin (Invitrogen Corporation, Carlsbad, CA, USA). Cells between 19 and 27 passages were used. Rat liver myofibroblasts (MFs) established previously [20] and rat hepatoma cell collection H4IIE (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM made up of 10% FBS. All cells were cultured at 37C in a humidified atmosphere made up of 5% CO2. For inducing bi-potential differentiation, WB-F344 cells were cultured in a medium Amuvatinib hydrochloride made up of 5 mM SB (Sigma-Aldrich, St. Louis, MO, USA) for 1 to 5 days. Immunoprecipitation and electrophoresis As previously explained, the Ligab antibody reacts specifically with the Ligab antigen in a non-denaturing protein extraction buffer [17]. Therefore, we prepared Lig-8 cell protein extracts by dounce-homogenizing the cells in a non-denaturing protein lysis buffer made up of 1% v/v Triton X-100, 50 mM Tris (pH 7.4), 300 mM NaCl, 5 mM EDTA, 0.02% w/v sodium azide, 1 mM phenylmethylsulfonyl fluoride, and 1% v/v protease inhibitor cocktail Amuvatinib hydrochloride (Sigma-Aldrich, St. Louis, MO, USA). The protein extracts were cleared by centrifugation at 12,000 at 4C for 30 minutes and the supernatants were further subjected to ultracentrifugation (Beckman Optima XL-90 Ultracentrifuge,.