Volume 117, Issue 3, Pages 669-677 (September 1999) Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas  Hideyuki Miyoshi, Christian Rust, Patricia J. Roberts, Lawrence J. Burgart, Gregory J. Gores  Gastroenterology  Volume 117, Issue 3, Pages 669-677 (September 1999) DOI: 10.1016/S0016-5085(99)70461-0 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Hepatocyte apoptosis in 3-day BDL mice is Fas dependent, but Fas ligand independent. TUNEL procedure was performed in livers obtained from 3 sham-operated controls and BDL WT, Fas-deficient lpr, and Fas ligand–deficient gld mice. The magnitude of apoptosis occurring in WT mice was greater than that in sham-operated control or BDL lpr mice (P < 0.01, ANOVA with a Bonferroni correction for multiple comparisons). In contrast, there was no significant difference between the magnitude of apoptosis occurring in BDL lpr vs. BDL gld mice (P = NS). Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Serum ALT values and liver caspase 3–like activity, but not serum bilirubin values, are greater in WT than lpr mice 3 days after BDL. WT and Fas-deficient lpr mice underwent a sham operation or ligation of their common bile duct. Three days after the surgical procedure, the mice were anesthetized, and blood was obtained for determination of serum ALT and total bilirubin. Liver tissue was procured for determination of caspase 3–like activity. Assays were performed as described in Experimental Procedures. (A) Serum ALT determinations. (B) Liver caspase 3–like activity. (C) Serum total bilirubin determinations. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Processing of procaspase 8 in BDL WT but not lpr mice. Three days after sham operation or ligation of the common bile duct in WT and lpr mice, liver tissue was obtained and processed for immunoblot analysis as described in Experimental Procedures. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride, and probed with an antibody recognizing both the zymogen and the 18-kilodalton processed subunit forms of caspase 8. The membranes were also probed with an antibody recognizing β-actin to assure equal protein loading in the respective lanes. The gels are representative of 3 experiments from 3 separate animals. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Animal survival is greater in BDL WT than in BDL lpr mice. Ten WT and lpr mice underwent BDL. Animal survival was monitored over 21 days. As assessed by contingency table analysis, the number of animals surviving in each group at the completion of the experiment was significantly different. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 After 3 days of BDL, apoptosis occurs in both WT and lpr mice but is greater in WT animals. TUNEL procedure was performed in livers obtained from WT and lpr mice 3, 7, 14, and 21 days after sham operation or ligation of the common bile duct. TUNEL-positive cells were visualized by fluorescence microscopy using excitation and emission wavelengths of 490 and 520 nm, respectively. The number of TUNEL cells per high-power field was quantitated at each time point. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Fas remains constant after BDL in WT mice and is not present in lpr mice. Liver tissue was obtained from WT and lpr mice 0, 3, 7, 14, and 21 days after ligation of the common bile duct and processed for immunoblot analysis. Proteins were separated by SDS–PAGE, transferred to polyvinylidene difluoride, and probed with an antibody recognizing Fas. The membranes were also probed with an antibody recognizing β-actin to assure equal protein loading in the respective lanes. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Immunoblot analysis of Bax after BDL. Liver tissue was obtained from a minimum of 3 WT and lpr mice each 0, 3, 7, 14, and 21 days after ligation of the common bile duct. Liver proteins were processed for immunoblot analysis. Proteins (50 μg/lane) were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with an antibody recognizing Bax. The membranes were also probed with an antibody recognizing β-actin to assure equal protein loading in the respective lanes. Comparisons between groups of animals were made by determining the ratio (protein of interest/β-actin) of the immunoreactive area by densitometry. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Mitochondrial-associated Bax increases in lpr but not WT mice after BDL. Mitochondria were obtained from a minimum of 3 WT and lpr mice each 7 days after ligation of the common bile duct and processed for immunoblot analysis. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with an antibody recognizing Bax. The membranes were also probed with an antibody recognizing cytochrome oxidase to assure equal loading in the respective lanes. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Immunoblot analysis of Bcl-xL after BDL. Liver tissue was obtained from a minimum of 3 WT and lpr mice each 0, 3, 7, 14, and 21 days after ligation of the common bile duct and processed for immunoblot analysis. Proteins (50 μg/lane) were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with an antibody recognizing Bcl-xL. The membranes were also probed with an antibody recognizing β-actin to assure equal protein loading in the respective lanes. Comparisons between groups of animals were made by determining the ratio (protein of interest/β-actin) of the immunoreactive area by densitometry. Although there was a trend for decreased Bcl-xL expression in BDL lpr mice, the data were not statistically significant.The difference between the day 0 and day 21 BDL lpr mice was P = 0.12 (n = 3 for each group). Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 10 Immunoblot analysis of Bcl-2 after BDL. Liver tissue was obtained from a minimum of 3 WT and lpr mice each 0, 3, 7, 14, and 21 days after ligation of the common bile duct and processed for immunoblot analysis. Proteins (50 μg/lane) were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with an antibody recognizing Bcl-2. The membranes were also probed with an antibody recognizing β-actin to assure equal protein loading in the respective lanes. Comparison between groups of animals was made by determining the ratio (protein of interest/β-actin) of the immunoreactive area by densitometry. Gastroenterology 1999 117, 669-677DOI: (10.1016/S0016-5085(99)70461-0) Copyright © 1999 American Gastroenterological Association Terms and Conditions