Volume 123, Issue 2, Pages 619-631 (August 2002) Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways  Edward M. Conway, Saskia Pollefeyt, Marta Steiner–Mosonyi, Wei Luo, Astrid Devriese, Florea Lupu, Francoise Bono, Nathalie Leducq, Frederique Dol, Paul Schaeffer, Désiré Collen, Jean–Marc Herbert  Gastroenterology  Volume 123, Issue 2, Pages 619-631 (August 2002) DOI: 10.1053/gast.2002.34753 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Inactivation of the Survivin gene in mice. The targeting vector, which has a neomycin gene replacing the region of the survivin gene that contains the 4 exons, was introduced into ES cells by homologous recombination as described in the Materials and Methods section. Southern blots of ES cell genomic DNA digested with (B) Kpn1/EcoRV or (C) Sac1/Spe1, or (D) tail DNA digested with Kpn1/EcoRV, hybridized with (B, D) 5'-external or (C) 3'-external probe, confirmed the expected genotype. Gastroenterology 2002 123, 619-631DOI: (10.1053/gast.2002.34753) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 H&E staining of liver sections. Histologic sections of livers from (A, C, D, G, H) survivin+/+ and (B, E, F, I, J) survivin+/− mice were stained with H&E. Normal architecture is preserved in livers from (A, B) sham-treated mice and from (C, D) survivin+/+ mice exposed to Jo2 500 ng/g. (E, F) Jo2 500 ng/g caused hemorrhagic necrosis of the liver in survivin+/− mice, which was more severe with (G, H, I, J) Jo2 1500 ng/g. Gastroenterology 2002 123, 619-631DOI: (10.1053/gast.2002.34753) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Detection of survivin in livers. Histologic sections of livers from (A, C, E, F) survivin+/+ and (B, D, G, H) surivivin+/− mice were immunoperoxidase stained with specific antisurvivin antibodies. Survivin is not detectable in the livers of (A, B) sham-treated mice or (C) survivin+/+ mice exposed to Jo2 500 ng/g. Survivin expression is observed in the cytoplasm of hepatocytes in survivin+/− mice exposed to (D, red arrows) Jo2 500 ng/g, and in the livers of all mice treated with (E, F, G, H) Jo2 1500 ng/g. Immunostaining of sections with preimmune immunoglobulin revealed no specific signal (not shown). Gastroenterology 2002 123, 619-631DOI: (10.1053/gast.2002.34753) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Detection of survivin in subcellular fractions. (A) After exposure to phosphate-buffered saline (sham) or Jo2 500 ng/g for 2 hours, mitochondrial (Mit) and nuclear (Nuc) fractions were isolated from the livers of different survivin+/− or survivin+/+ mice, and 100 μg of each were separated by SDS-PAGE for immunoblotting to detect survivin, identified as an ~16-kilodalton band. (B) Total liver lysates (100 μg/lane) from mice exposed to different doses of Jo2 were Western immunoblotted to detect survivin. Gastroenterology 2002 123, 619-631DOI: (10.1053/gast.2002.34753) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Western immunoblots of liver cell lysates. (A) Equal quantities (50 μg) of liver lysates, each from different survivin+/+ (+/+) or survivin+/− (+/−) mice that were treated with intraperitoneal phosphate-buffered saline (sham) or Jo2 500 ng/g, were separated by SDS-PAGE and Western immunoblotted for the proteins shown on the left. Molecular weight markers for each blot are on the right. Procaspase-3 is detected at ~32 kilodaltons, whereas activation is represented by generation of a ~17-kilodalton fragment. Activation of Bid is revealed by the presence of a tBid fragment of ~15 kilodaltons. A caspase-8 activation fragment is detected as an ~20-kilodalton band. Procaspase-9 and activated products of caspase-9 are detected as proteins at ~46 kilodaltons and ~18 kilodaltons, respectively. (B) Equal quantities of cytosol (50 μg) from livers were separated by SDS-PAGE and Western immunoblotted for detection of cytochrome c, revealed by a prominent band at ~15 kilodaltons and dimers at ~30 kilodaltons. Gastroenterology 2002 123, 619-631DOI: (10.1053/gast.2002.34753) Copyright © 2002 American Gastroenterological Association Terms and Conditions