Volume 128, Issue 5, Pages 1405-1415 (May 2005) Cystathionine β Synthase Deficiency Promotes Oxidative Stress, Fibrosis, and Steatosis in Mice Liver  Karine Robert, Johnny Nehmé, Emmanuel Bourdon, Gérard Pivert, Bertrand Friguet, Claude Delcayre, Jean–Maurice Delabar, Nathalie Janel  Gastroenterology  Volume 128, Issue 5, Pages 1405-1415 (May 2005) DOI: 10.1053/j.gastro.2005.02.034 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Detection of oxidatively modified cytosolic proteins. Cytosolic extracts were prepared from liver of 3-month-old CBS +/+ mice (+/+) and CBS −/− mice (−/−). The coomassie blue stained gel of total proteins is presented in A. The detection of oxidatively modified proteins (B) was accomplished using the Oxyblot-kit. The extracts were treated with DNPH to derive protein carbonyls and then separated by SDS-PAGE (10 μg protein per lane, 12% polyacrylamide). Western blot analysis was performed utilizing polyclonal antibodies to the 2, 4-dinitrophenyl moiety and antibody for SOD1 (SOD1) as control hybridization.7 For quantification, the optical densities of 2, 4-dinitrophenyl moiety immunoreactive bands were measured and normalized to the optical densities of SOD1 immunoreactive bands in the same samples. Gastroenterology 2005 128, 1405-1415DOI: (10.1053/j.gastro.2005.02.034) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Histopathologic changes in the liver of CBS −/− mice. Liver specimens were taken from mouse livers of 8-, 18-, and 32-week-old CBS +/+ (+/+) and CBS −/− (−/−) mice for microscopic examination. Microtome-generated sections prepared from the excised liver specimens were stained with either H&E (A) to assess liver histology or Sirius red to assess development of liver fibrosis (B). Photomicrographs from livers of 8-week-old CBS +/+ and −/− mice were more contrasted to show mild fibrosis around the vessels. Cryostat sections prepared from the excised liver specimens were stained with either oil red O counterstained with hematoxylin (C) or Nile red (D) to assess steatosis. The results are representative photomicrographs from 3 independent experiments. Scale bars = 100 μm. Gastroenterology 2005 128, 1405-1415DOI: (10.1053/j.gastro.2005.02.034) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Expression of TIMP-1 and TNF-α in liver of CBS −/− mice. (A) Western blot analysis of TIMP-1 expression. Liver extracts prepared from CBS +/+ (+/+) and CBS −/− (−/−) mice were separated by a 12% SDS-PAGE and immunoblotted with an anti–TIMP-1 antibody (TIMP-1). (B) Expression of TNF-α (TNF-α)μ in liver extracts prepared from CBS +/+ (+/+) and CBS −/− (−/−) mice. An antibody for SOD1 (SOD1) is used as control hybridization.7 For quantification, the optical densities of TIMP-1 and TNF-α immunoreactive bands were measured and normalized to the optical densities of SOD1 immunoreactive bands in the same samples. Gastroenterology 2005 128, 1405-1415DOI: (10.1053/j.gastro.2005.02.034) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Analysis of proapoptotic, antiapoptotic proteins, DNA laddering, and TUNEL positive cells in liver of CBS +/+ and CBS −/− mice. (A) Western blot analysis of Bax and Bcl-2 expression. Liver extracts prepared from CBS +/+ (+/+) and CBS −/− (−/−) mice were separated by a 12% SDS-PAGE, immunoblotted with an anti-Bax antibody (Bax), stripped, and immunoblotted with an anti–Bcl-2 antibody (Bcl-2). An antibody for SOD1 (SOD1) is used as control hybridization.7 For quantification, the optical densities of Bax and Bcl-2 immunoreactive bands were measured and normalized to the optical densities of SOD1 immunoreactive bands in the same samples. (B) Agarose gel analysis of genomic DNA. Genomic DNA was isolated from liver of 12-, 16-, and 20-week-old CBS +/+ (+/+) and CBS −/− (−/−) mice and analyzed for DNA fragmentation. CON, control DNA. (C) Photomicrographs of TUNEL-positive brown nuclei (arrows) within cells from CBS +/+ (+/+) and CBS −/− (−/−) mouse livers. Microtome-generated sections prepared from the excised liver specimens were stained with TUNEL to show apoptotic cells. The results are representative photomicrographs from 3 independent experiments. Scale bars = 50 μm. Gastroenterology 2005 128, 1405-1415DOI: (10.1053/j.gastro.2005.02.034) Copyright © 2005 American Gastroenterological Association Terms and Conditions