9 ROS formation was measured using a multiwell fluorescence scanner (CytoFluor 2300; Millipore, Bedford, MA). Liver extracts were obtained in a modified radioimmunoprecipitation buffer as described.18 Western blotting was performed using standard protocols. An antibody against αSMA (Sigma-Aldrich) was used at the concentration of 1:1000. Horseradish peroxidase–conjugated secondary antibodies were used and visualized with enhanced chemiluminescence. Bone marrow transplantation (BMT) was performed as described.25 Mice received an intravenous injection

of liposomal clodronate (200 μL intravenously) before irradiation to deplete KCs.27 Tibias and femurs of donor mice were Ivacaftor clinical trial flushed to obtain bone marrow (BM). BM cells (1 × 107) were injected into

the tail veins of lethally irradiated (11 Gy) recipient mice. BDL was performed 12 weeks after BMT. To determine successful BMT in p47phox-deficient and p47phox-sufficient mice, spleen cells were isolated from BDL chimeric mice and analyzed by quantitative real-time polymerase chain reaction (RT-PCR) to measure p47phox messenger RNA (mRNA) expression (data not shown). RT-PCR was used for measuring mRNA levels of fibrogenic markers (collagen α1(I) and αSMA). Extraction of RNA from total liver of mice was performed by a combination of TRIzol (Invitrogen, Carlsbad, CA) and RNeasy columns (Qiagen, Valencia, CA). Complementary DNA was obtained using the Amersham Deforolimus chemical structure kit for complementary DNA synthesis.11 Results are expressed as mean ± standard error of the mean. The results were analyzed using RVX-208 the unpaired Student t test or the Newman-Keuls test. A P value < 0.05 was considered statistically significant. To assess the role of the NOX in liver fibrosis, p47phox knockout (KO) mice were subjected to two different models of hepatic damage: BDL as a model of cholestatic liver injury and CCl4 treatment as a model of toxic liver injury. Consistent with our previous studies,9 mice deficient for the p47phox component of NOX had reduced fibrosis after 3 weeks of BDL, as evaluated by collagen deposition and αSMA staining (Fig. 1A,B). Furthermore, the

critical role of NOX in liver fibrosis was confirmed in mice subjected to intraperitoneal injection of CCl4. Mice received 16 injections of CCl4 (0.5 μL/g body weight; twice weekly) and were sacrificed 2 days after the last injection. WT mice displayed a significant increase in collagen deposition and αSMA staining following treatment with CCl4 compared to vehicle-treated mice (Fig. 1C,D). The increase in fibrotic parameters was significantly reduced in p47phox-deficient mice (Fig. 1C,D). In addition, mRNA levels of collagen α1(I) and αSMA were significantly reduced in p47phox-deficient mice compared to p47phox-sufficient mice either after 3 weeks of BDL or after 16 injections of CCl4, as evaluated by RT-PCR (Fig. 1E,F).