Inactivation of Fah leads to an accumulation of toxic metabolites, such as fumarylacetoacetate (FAA), which subsequently causes

acute or chronic liver injury.[8] ICG-001 datasheet HT1 is characterized by an extremely high susceptibility to liver cancer. A murine model of Fah deficiency has been developed that represents all phenotypic and biochemical manifestations of the human disease on an accelerated time scale.[9-12] C57Bl6-FahDexon5 and C57Bl6-Cdkn1atm1Ty1/J mice were crossed to generate Fah+/−/p21+/− breeders from which all Fah−/− and Fah−/−/p21−/− animals used in these studies were derived. Drinking water was supplemented with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) at a concentration of 7.5 mg/mL; 2.5% percent of this normal dose was used for low-dose NTBC treatment. Ten-week-old Fah-deficient mice were monitored for survival after NTBC was reduced (2.5%) or withdrawn (0%). Fah−/− and Fah/p21−/− double-knockout mice on 2.5% NTBC were followed for 400 days and Fah/p21−/− 0% NTBC for 90 days. Briefly, mice were injected intraperitoneally with a ketamine (100 mg/kg body weight)/xylazine (4 mg/kg body weight) solution and subjected to a midline incision.

The left and median lobes of the liver were ligated and resected. After closing the peritoneal and skin wounds, mice recovered from anesthesia on a warming pad. Thirty-eight hours or 1 week after PH, mice were sacrificed and livers were collected. Frozen liver tissue was homogenized using an ultraturax http://www.selleckchem.com/products/Gefitinib.html (10 seconds) in cell lysis buffer [50 mM 4-(2-hydroxyethyl)−1-piperazine

ethanesulfonic acid, 50 mM potassium chloride, 50 mM sodium fluoride, 5 mM tetrasodium diphosphate decahydrate, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 5 mM β-glycerophosphate, Parvulin 1 mM dithiothreitol, 1 mM vanadate, 1% (vol/vol) Nonidet P40] containing a Complete Protease Inhibitor mixture (Roche) and centrifuged for 10 minutes at 16,000g. Protein concentration was measured using the Bio-Rad Protein Assay Dye Reagent, and equal amounts of protein extracts were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted to activated-polyvinylidene difluoride membrane (Bio-Rad). Mouse blood was collected from the orbital sinus in lithium heparin tubes (LH1.3, Sarstedt, Germany) and processed according to the manufacturer’s instructions. Aminotransferase and bilirubin levels were measured using an Olympus AU 400 system (Beckman Coulter, Switzerland). Total RNAs from liver tissue (n = 4) were extracted using the Qiagen RNAeasy kit. A Transcriptor High Fidelity cDNA Synthesis kit (Roche) was used to synthetize complementary DNA. Sequences of polymerase chain reaction (PCR) primers are provided upon request. Data are presented as the mean ± SD. Data were analyzed via analysis of variance followed by a Student t test to determine significance. P values were considered statistically significant at P < 0.05, P < 0.01, or P < 0.