1. Epidermal Growth Factor Receptor–Mediated Proliferation of Enterocytes Requires p21waf1/cip1 Expression 
George Sheng, Kathryn Q. Bernabe, Jun Guo, Brad W. Warner 
Gastroenterology 
Volume 131, Issue 1, Pages (July 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Western blot of total EGFR protein expression and activity in IEC-6 cells after treatment with EGF for various times. Immunoprecipitation (IP) of the EGFR was performed with subsequent immunoblotting (IB) for total (nonphosphorylated) or activated (phosphotyrosine) EGFR.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
(A) Real-time PCR for p21waf1/cip1 mRNA expression in IEC-6 cells stimulated with EGF (100 ng/mL) at various time points. Values are expressed as fold differences relative to unstimulated cells at 0 hours. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs 0 hour time point. (B) Western blots of p21waf1/cip1 protein expression in IEC-6 and HCA-7 cells stimulated with EGF (100 ng/mL) at various time points. Actin served as a loading control. The graphs below the blots represent corresponding fold differences in relative densitometric readings relative to unstimulated cells at 0 hours. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs 0 hour time point. (C) Western blots of p21waf1/cip1 protein expression in IEC-6 cells at various times in response to EGF (100 ng/mL) stimulation in the absence (left panel) or presence (right panel) of the selective EGFR inhibitor (ZD1839). Actin was used to ensure equal loading.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
p21 Promoter activity in HCA-7 cells in response to EGF. Cells were cotransfected with either the 2.4-kb full-length human P21waf1/cip1 promoter or several truncations of the promoter and SV-40 promoter-driven renilla luciferase plasmids. The final construct SMAΔ2 represents the full-length p21 promoter minus the 62 base promoter region present in the SMAΔ1 construct. Following transfection, cells were stimulated with EGF (100 ng/mL) for 2 hours, and resulting luciferase activity was compared with cells without EGF treatment. The graph represents fold differences in luciferase activity relative to unstimulated cells. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs unstimulated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Western blot demonstrating the effects of PI3-K or MAPK inhibition on EGF-stimulated P21waf1/cip1 protein expression in IEC-6 and HCA-7 cells. Cells were pretreated with either the PI-K inhibitor LY (25 nmol/L) or the MAPK inhibitor UO126 (20 nmol/L) and then stimulated with EGF (100 ng/mL). Inhibition of the PI3-K and MAPK pathways was verified by measuring phosphorylated (P)-AKT and ERK, respectively. Actin was used to demonstrate equal loading between lanes.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
The effect of PI3-K or MAPK inhibition on p21 promoter activity in IEC-6 cells treated with EGF. Cells were cotransfected with either the 2.4-kb full-length human P21waf1/cip1 promoter or SV-40 promoter-driven renilla luciferase plasmids. Following transfection, cells were pretreated with either the PI-K inhibitor LY or the MAPK inhibitor UO126 and then stimulated for 1 hour with EGF (100 ng/mL). The resulting luciferase activity was compared with cells without EGF treatment. The graph represents fold differences in luciferase activity relative to unstimulated cells. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs unstimulated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
(A) Western blot of p21waf1/cip1 protein expression in HCA-7 cells in response to transfection with plasmids containing constitutively active H-Ras, Raf, or MEK-1. Phosphorylated ERK served as a marker for MAPK activity. Control represents nontransfected cells. (B) p21waf1/cip1 Promoter activity in HCA-7 cells in response to transfection with plasmids containing constitutively active H-RAS, Raf, or MEK-1. In addition, cells were cotransfected with the 2.4-kb full-length human P21waf1/cip1 promoter and SV-40 promoter-driven renilla luciferase plasmids. The values represent fold changes in luciferase (LUC) activity relative to nontransfected cells after normalization to renilla luciferase. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
Effect of p21 silencing on toxicity of IEC-6 cells. Apoptosis in IEC-6 cells was determined using an apoptosis detection ELISA kit (Roche, Indianapolis, IN). Control cells were grown in 10% FBS; X-treme gene (Roche, Indianapolis, IN) was the transfection reagent; and Sicon was the scrambled control RNA sequence. Experiments were performed in triplicate. There were no statistically significant differences between groups.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

9. Figure 8
Effect of EGF stimulation of proliferation and p21waf1/cip1 protein expression in IEC-6 and HCA-7 cells following p21waf1/cip1 RNA silencing. P21 siRNA and siControl (scrambled) constructs were transfected into cells. After confirmation of attenuated p21waf1/cip1 expression by Western blotting, cells were stimulated with EGF (100 ng/mL), and proliferation was measured using a BrdU ELISA Assay Kit (Roche). A proliferation index was derived as the percentage of proliferating cells; *denotes P < .05 EGF-stimulated vs nonstimulated cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

10. Figure 9
Western blot demonstrating the effect of p21waf1/cip1 silencing on p21waf1/cip1, Cdk4, and cyclin D1 protein expression in IEC-6 cells in response to EGF. P21waf1/cip1 expression was silenced by transfecting cells with p21waf1/cip1 siRNA (Dharmacon, Lafayette, CO), and control cells were transfected with siCONTROL (nontargeting siRNA; Dharmacon). Cells were then stimulated with EGF (100 ng/mL) for various times.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

11. Figure 10
Western blot demonstrating the effect of p21waf1/cip1 silencing on protein interactions between p21waf1/cip1 and Cdk4 and cyclin D1 with Cdk4 in response to EGF in IEC-6 cells. P21waf1/cip1 expression was silenced by transfecting cells with p21waf1/cip1 siRNA, and control cells were transfected with siCONTROL (scrambled siRNA). Cells were then stimulated with EGF (100 ng/mL) for various times. Immunoprecipitation was then done with an anti-Cdk4 antibody, and expression of Cdk4, cyclin D1, and p21waf1/cip1 was determined.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

12. Figure 11
(A) Western blot demonstrating p21waf1/cip1 levels in small intestine mucosa of mice with different levels of EGFR activity: wild-type controls (WT) with normal EGFR function, EGF transgenic mice with elevated EGF expression in intestinal mucosa,14 and Wave-2 mice with perturbed EGFR activity. Three animals from each group were used for Western blot analysis, quantitatively measured by densitometry, and the results were graphed as fold of changes compared with WT mice. *denotes P < .05 compared with WT. (B) Enterocyte proliferation in Control (C57/Bl6) or p21waf1/cip1-null mice at 3 days following a 50% proximal small bowel resection (SBR) or sham operation (division of the bowel with reanastomosis alone). A quantitative index of proliferation was derived by subcutaneous administration of 5-bromodeoxyuridine (BrdU) to mice 1 hour prior to death. Incorporation of BrdU into proliferating crypt cells was detected using immunohistochemistry and an index calculated as the percentage of crypt cells that are BrdU positive. One set of p21waf1/cip1-null mice were given 50 μg/kg/day of human recombinant EGF (Sigma Chemical Co, St. Louis, MO) via twice daily orogastric gavage. N = 2 per group; *denotes P < .05 SBR vs sham.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions