In hepatocytes, NOX4 causes cell death but does not mediate epithelial-mesenchymal transition (EMT). These results open new perspectives for the involvement of NOXes in liver fibrosis and for the potential development of new therapeutic targeted tools. Materials and Methods Ethics statement Mice were housed in accordance with European laws and with the general regulations selleck chem inhibitor specified by the Good Scientific Practices Guidelines of the Medical University of Vienna. From Spain, the approval for all the experiments related to the study of liver fibrosis in experimental animal models was applied to the General Direction of Environment and Biodiversity, Government of Catalonia, and approved with the number #4589, 2011 (document enclosed).
Human tissues were collected with the required approvals from the Institutional Review Board (Comit�� ��tico de Investigaci��n Cl��nica del Hospital Universitario Fundaci��n Alcorc��n) and patient’s written consent conformed to the ethical guidelines of the 1975 Declaration of Helsinki (both documents are enclosed). Reagents and antibodies TGF-�� was from Merck (Darmstadt, Germany). Fetal bovine serum was from Sera Laboratories International (Cinder Hill, UK). Glutathione-ethyl-ester (GEE), Diphenyleneiodonium chloride (DPI) and Butylated hydroxyanisole (BHA) were from Sigma (St Louis, USA). The caspase-3 substrate Ac-DEVD-AMC was from Pharmingen (San Diego, CA, USA).
Antibodies: mouse anti-��-actin (clone AC-15, Sigma), rabbit anti-cleaved caspase-3 (Asp-175) from Cell Signaling Technology (Danvers, MA, USA), anti-F4/80 (Abcam, Cambridge, UK), mouse anti-E-cadherin (BD Pharmingen, NJ, USA), rabbit anti-ki67 (Abcam), mouse anti-NOX2 (Santa Cruz Biotechnology, CA, USA), anti-NOX4 raised by Sigma-Genosys against a peptide corresponding to the C-terminal loop region (aminoacids 499�C511), mouse anti-��-SMA (Sigma, St Louis, USA), rabbit anti-phospho-Smad2 (Ser465/467) and rabbit anti-phospho-Smad3 (Ser423/425) from Cell Signaling Technology, goat anti-Smad2/3, anti-Smad7 and anti-TGF-�� from Santa Cruz Biotechnology and mouse anti-vimentin (Sigma, St Louis, USA). Mice Three animal experimental models of liver fibrosis were used for this study: two genetically modified mice and one drug-induced model.
Mdr2?/?/p19ARF?/? double null mice  displayed a fibrotic phenotype comparable to Mdr2?/? mice, widely used as a model for experimental liver fibrosis , , characterized by severe hepatic injury and large periductal accumulation of MFBs, but GSK-3 showed the additional advantage of allowing the isolation of immortal cells for in vitro experiments . Stat3��hc/Mdr2?/? mice show Stat3 conditional inactivation specifically in hepatocytes and cholangiocytes in a Mdr2?/? background , which strongly aggravates liver injury and fibrosis.