Our data show that chronic hepatocellular NF-κB activation is sufficient for liver fibrosis development by way of

recruitment and activation of macrophages. α-SMA, alpha-smooth muscle actin; ALT, alanine amino transferase; AST, aspartate amino transferase; CA, constitutively active; CT, control; DOX, doxycycline; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; HPF, high-power field; HPRT, hypoxanthine-guanine phosphoribosyltransferase gene; HSC, hepatic stellate cell; IKK, IκB kinase; LAP, liver activator protein; LPS, lipopolysaccharide; NASH, nonalcoholic steatohepatitis; Torin 1 in vivo NF-κB, nuclear factor-κB; selleck kinase inhibitor PDGF, platelet-derived growth factor; RLU, relative light unit; SAA, serum amyloid A; TGF, transforming growth factor; tTA, tetracycline transactivator. We crossed mice carrying

a constitutively active human IKK2 (CAIKK2) allele17 under the control of a tissue-specific tetracycline-inducible system with animals expressing tetracycline-responsive transactivator (tTA) under the control of the rat liver activator protein (LAP) promotor.14 The generated mice were on a C57BL/6 and NMRI mixed background and were backcrossed at least four times to a C57BL/6 background. Studies were performed on male mice kept under specific pathogen-free conditions. The experiments were approved by the State of Baden-Württemberg in Germany and the University of Ulm Animal Care Committee. To avoid the embryonic activation of the IKK2/NF-κB system, all mice received 0.1 g/L doxycycline in drinking water until birth. In some cases, 4-week-old animals were readministered 0.1 g/L doxycycline (DOX) in drinking water for

3 days, or 12-week-old animals were readministered DOX for 3 weeks, to study whether a continuous CAIKK2 expression is required for the observed liver phenotype. Of note, CAIKK2 mice contain a bidirectional promoter, whose activation leads to simultaneous production of both IKK2 and Photinus pyralis luciferase.17 Mice were sacrificed by way of CO2 inhalation and blood was collected from vena cava inferior. After brief centrifugation, serum was collected and used for measurement of alanine and aspartate aminotransferase levels (ALT and AST; Reflotron Histamine H2 receptor system, Roche). Livers were removed, weighed, and divided into pieces that were fixed in 10% formaldehyde for histological/immunohistochemical analysis, snap-frozen in liquid nitrogen for molecular or biochemical analysis, or rapidly frozen for immunofluorescence staining. For preparing whole liver extract, frozen livers were lysed in Dignam C buffer18 or in RIPA buffer.19 The lysate was centrifuged at 20,000g for 30 minutes at 4°C and the supernatant was recovered. Protein extracts were electrophoresed and subsequently blotted.