Volume 130, Issue 2, Pages 412-423 (February 2006) Epidermal Growth Factor Receptor Signaling Regulates Bax and Bcl-w Expression and Apoptotic Responses During Intestinal Adaptation in Mice  Nicole P. Bernal, Wolfgang Stehr, Rebecca Coyle, Christopher R. Erwin, Brad W. Warner  Gastroenterology  Volume 130, Issue 2, Pages 412-423 (February 2006) DOI: 10.1053/j.gastro.2005.11.006 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 Average ± SEM mRNA expression of Bax, Bcl-w, and the ratio of expression of Bax to Bcl-w at 3 and 7 days after a 50% proximal small-bowel resection (SBR) or sham (transection and reanastomosis). The mRNA expression was measured by quantitative RT-PCR in enterocytes from the villus tips (A) and crypts (B) collected in the remnant ileum by laser capture microdissection. The bars represent average n-fold change in expression in SBR mice vs sham-operated animals. The determination of mRNA expression from SBR and sham-operated mice was run simultaneously for each reaction as animals became available, and all results were pooled (n = 10 mice per group). RT-PCR was run in triplicate for each specimen. *P < .05, Bax vs Bcl-w expression. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 Average ± SEM mRNA expression of Bax (A), Bcl-w (B), and the Bax–Bcl-w ratio (C) as measured by quantitative real-time RT-PCR in enterocytes isolated from the crypt and villus tips in the remnant ileum by laser capture microdissection in control, waved-2, and transgenic EGF-overexpressing mice (EGFtg) at 3 days after either 50% proximal small-bowel resection (SBR) or sham (transection and reanastomosis) operation. The bars represent the n-fold change in expression in each strain after SBR when compared with sham-operated animals. The mRNA expression from SBR and sham-operated mice was run simultaneously for each reaction as animals became available, and all results were pooled (n = 6–12 mice per group). RT-PCR was run in triplicate for each specimen. *P < .05, crypt vs villus expression of Bax for each group; #P < .05, Bcl-w crypt expression vs waved-2 Bcl-w crypt expression. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 Adaptive changes in the remnant ileum at 3 days after 50% proximal small-bowel resection (SBR) or sham operation (transection and reanastomosis only) in control (C57BL/6), Bax-null, EGF-treated (EGF), or Bax-null mice treated with EGF. EGF treatment consisted of twice-daily orogastric EGF (50 μg/kg per day) or saline vehicle. Adaptive morphological parameters consisted of villus height (A) and crypt depth (B). Alterations in enterocyte kinetics were recorded as indices of proliferation (C) and apoptosis (D). A proliferative index was derived by counting the number of positively labeled BrdU crypt cells divided by the total number of crypt cells. An apoptotic index was derived from number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling–positive cells per crypt and was confirmed by abnormal morphology. Values are mean ± SEM; n = 5–7 per group. *P < .05, sham vs SBR; #P < .05, SBR vs all other SBR; @P < .05, EGF SBR vs Bax-null and control SBR. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 Protein expression of Bax (A) and Bcl-w (B) in Bax-null and control mice 3 days after 50% proximal small-bowel resection (SBR) or sham operation (transection and reanastomosis only) and randomized to receive orogastric saline or EGF. The remnant ileum was harvested from 3–8 animals per group and then pooled according to mouse strain and treatment group. Numerical values of Bax and Bcl-w band density after normalization to actin protein loading are represented by the bar graph above the corresponding Western blot. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 5 Percentage of healthy survivors in control (C57BL/6), Bcl-w–null, transgenic EGF-overexpressing (EGFtg), and Bcl-w–null mice crossed with EGFtg mice (Bcl-w–null EGFtg) at 3 days after a 50% proximal small-bowel resection (SBR; n = 112; 12–50 per group) or sham operation (transection and reanastomosis only; n = 52; 8–14 per group). *P < .05, sham vs SBR; #P < .05, SBR vs all other SBR; @P < .05, SBR vs Bcl-w–null and Bcl-w–null × EGFtg SBR; §P < .05, sham vs control sham. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 6 Adaptive changes in the remnant ileum at 3 days after 50% proximal small-bowel resection (SBR) or sham operation (transection and reanastomosis only) in control (C57BL/6), Bcl-w–null, transgenic EGF-overexpressing mice (EGFtg) or Bcl-w–null mice crossed with EGFtg mice. Adaptive morphological parameters consisted of villus height (A) and crypt depth (B). Alterations in enterocyte kinetics were recorded as indices of proliferation (C) and apoptosis (D). A proliferative index was derived by counting the number of positively labeled BrdU crypt cells divided by the total number of crypt cells. An apoptotic index was derived from the number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling–positive cells per crypt and was confirmed by abnormal morphology. Values are mean ± SEM. Sham-operated animals had an n of 52 (8–14 per group). SBR animals had an n of 112 (12–50 per group). *P < .05, sham vs SBR; #P < .05, EGFtg SBR vs all other SBR; @P < .05, SBR vs Bcl-w knockout SBR; §P < .05 for Bcl-w knockout sham vs all other sham groups. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 7 Protein expression of Bcl-w (A) and Bax (B) in the remnant ileum at 3 days after a 50% proximal small-bowel resection (SBR) or sham operation (transection and reanastomosis only) from control (C57BL/6), Bcl-w–null, transgenic EGF-overexpressing mice (EGFtg) and Bcl-w–null crossed with EGFtg mice after 50% SBR or sham operation. The remnant ileum was harvested from 3–8 animals per group and then pooled according to mouse strain and treatment group. (A) Numerical values of Bcl-w and Bax band density after normalization to actin protein loading are represented by the bar graph above the corresponding Western blot. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 8 Effects of intestinal resection on apoptotic responses in the context of impaired, normal, or enhanced EGFR signaling capacity. EGFR, epidermal growth factor receptor. Arrows correspond to the relative expression of Bax or Bcl-w protein within the remnant ileum. NC, no change. Gastroenterology 2006 130, 412-423DOI: (10.1053/j.gastro.2005.11.006) Copyright © 2006 American Gastroenterological Association Terms and Conditions