Volume 135, Issue 2, Pages 539-551.e3 (August 2008) Raf Protects Against Colitis by Promoting Mouse Colon Epithelial Cell Survival Through NF-κB  Karen L. Edelblum, M. Kay Washington, Tatsuki Koyama, Sylvie Robine, Manuela Baccarini, D. Brent Polk  Gastroenterology  Volume 135, Issue 2, Pages 539-551.e3 (August 2008) DOI: 10.1053/j.gastro.2008.04.025 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Raf KOIE mice exhibit increased crypt injury and inflammation resulting from DSS colitis. Epithelial and stromal fractions were isolated from the (A) colon and small intestine of oil- or tamoxifen-injected Rafflx/flx;villin-Cre mice. Western blot analysis was performed with antibodies against Raf-1, B-Raf, E-cadherin, and actin. (B) H&E staining of colon sections from wild-type and Raf KOIE mice treated with water, 3% DSS for 4 days, or 4 days plus a 3-day recovery period. Scale bar, upper 2 rows, 500 μm or lower 2 rows, 125 μm. Insets, yellow boxes designate regions of ulceration. (C) Combined scores grading severity of inflammation and extent of crypt damage in wild-type or Raf KOIE mice following DSS treatment. Horizontal lines represent the mean. (D) Crypt height in wild-type and Raf KOIE mice following DSS treatment. Horizontal lines represent the median value. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Loss of Raf expression results in worsened clinical signs of colitis. (A) H&E staining of wild-type and Raf KOIE mice following 8 day DSS treatment. Scale bars, upper row, 500 μm or lower row, 250 μm. (B) Graphical representation of the average percent of weight loss for wild-type and Raf KOIE mice compared with the weight at the start of DSS treatment. (C) Colon length is represented as a ratio (cm:g) compared with the starting weight of mice prior to DSS administration. Horizontal bars represent the median value. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Raf protects from apoptosis in response to damage in the intestinal epithelium. (A) Apoptotic cells were detected by in situ oligo ligation (ISOL) in colon sections of DSS-treated wild-type and Raf KOIE mice following 1 or 4 days injury or 3 days recovery. Scale bar, 50 μm. (B) The average number of ISOL-positive cells was represented graphically. Horizontal lines represent the median. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Raf is required for the epithelial hyperproliferative response following DSS-induced injury. (A) Proliferating cells were detected by Ki67 and phospho-histone H3 staining in colon sections of wild-type and Raf KOIE mice during 3-day recovery from DSS-induced colitis. Scale bars, 125 μm. (B) The percentage of Ki67-positive cells in the midcolon is represented graphically. (C) The number of cells per hemicrypt is represented graphically. (D) Phospho-ERK staining of colon sections from control and Raf KOIE mice during 3-day recovery. Scale bars, 125 μm. (E) The average number of phospho-ERK-positive cells per crypt is represented graphically. Horizontal lines represent the median. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Raf is required for intestinal epithelial cell survival in response to DSS. YAMC cells were transfected with nontargeting or Raf siRNA and treated with 3% or 5% DSS for 7 hours. (A) Apoptosis was determined by TUNEL assay (scale bars, 100 μm), and (B) the percent of TUNEL-positive cells was assessed. (C) Transfected cells were treated with IL-13 (10 ng/mL) or 5% high (500 kilodaltons) or low (36–50 kilodaltons) molecular weight DSS for 7 hours, and the number of apoptotic cells was quantified by TUNEL assay. (D) Nontargeting or Raf siRNA-transfected YAMC cells were treated with 5% DSS for the times indicated. Western blots with whole cell lysates from DSS- or TNF (100 ng/mL, 15 minutes)-treated siRNA-transfected cells were probed for Raf, B-Raf, phospho-ERK, IκB, phospho-Akt, phospho-p38, phospho-JNK, and actin antibodies. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 MEK inhibitors protect against DSS-induced apoptosis. YAMC cells were treated with MEK inhibitors, PD98059 (20 μmol/L) or U0126 (10 μmol/L), and 5% DSS for 7 hours. (A) Apoptosis was detected by TUNEL assay, and (B) whole cell lysates were blotted with antiphospho-ERK, IκB, and actin. (C) YAMC cells were transfected with nontargeting or Raf siRNA and treated with U0126 (10 μmol/L) and 5% DSS for 7 hours. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 NF-κB activation rescues DSS-induced apoptosis in the absence of Raf expression. (A) Western blot analysis was performed on NF-κB p65, Raf, or IκBα siRNA-transfected YAMC cells to detect Raf, IκB, NF-κB p65, and actin expression. (B) NF-κB p65 nuclear translocation was detected in untreated or 5% DSS-treated (2 hours) nontargeting, IκBα, or Raf siRNA-transfected cells by immunocytochemistry. Scale bars, 20 μm. (C) Apoptosis in 5% DSS-treated p65, Raf, and/or IκBα siRNA-transfected YAMC cells was assessed by TUNEL assay. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Raf promotes NF-κB activation in the colon epithelium in response to DSS-induced injury. (A) Representative images are shown of immunofluorescence for NF-κB p65 (green), E-cadherin (red), and DAPI-stained nuclei (blue) in colon sections of wild-type and Raf KOIE mice following water treatment or DSS-induced injury. Scale bars, 20 μm. (B) The number of NF-κB p65-positive cells is represented graphically. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 Representative images of specific injury and inflammation scores. Regions representing the most severe damage in colon sections of a specific injury/inflammation score are shown. The histology was scored according to severity and extent of crypt damage and inflammation as well as percent colon involvement. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 Decreased cell proliferation during DSS-induced injury is not dependent upon Raf expression. The percentage of Ki67-positive cells following 1–3 days DSS treatment are represented graphically. Horizontal bars represent the median value. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 3 Raf expression does not affect maintenance of an Lgr5-positive cell population in the colon epithelium. In situ hybridization was performed for Lgr5 in colon sections of wild-type and Raf KOIE mice following water treatment and 3 day recovery following DSS-induced injury. Darkfield images show Lgr5 (orange) and eosin (green). Scale bars = 500 or 100 μm. Gastroenterology 2008 135, 539-551.e3DOI: (10.1053/j.gastro.2008.04.025) Copyright © 2008 AGA Institute Terms and Conditions