1. Recruitment of Ataxia-Telangiectasia Mutated to the p21waf1 Promoter by ZBP-89 Plays a Role in Mucosal Protection 
Longchuan Bai, John Y. Kao, David J. Law, Juanita L. Merchant 
Gastroenterology 
Volume 131, Issue 3, Pages (September 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

2. Figure 1
Butyrate induction of p21waf1 gene expression requires ZBP-89. (A) HCT116 cells were first transfected with a mutant (Mut) or wild-type (WT) siRNA oligonucleotide of ZBP-89 for 2 days, then treated with 2.5 mmol/L sodium butyrate for 18 hours. Immunoblotting was performed to determine the expression of ZBP-89, p21waf1, H2A, and Ac-H2A. β-tubulin was used as a loading control. (B) HCT116 p53 (−/−) cells were transfected with a WT or Mut ZBP-89 siRNA oligonucleotide for 2 days, then treated with 2.5 mmol/L sodium butyrate or 0.2 μmol/L TSA for 18 hours. Immunoblotting was performed as described earlier and glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. (C) A549 and HeLa cells were transfected with a WT or Mut ZBP-89 siRNA oligonucleotide for 2 days, then treated with 2.5 mmol/L butyrate or 0.2 μmol/L TSA for 18 hours. Immunoblotting was performed as described earlier.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

3. Figure 2
Butyrate recruits ZBP-89 to the GC-rich element of the p21waf1 proximal promoter. (A) HCT116 cells were treated with 2.5 mmol/L sodium butyrate for 1 hour and soluble chromatin was prepared. ChIP assays were performed using rabbit anti–ZBP-89 IgG and the normal rabbit IgG was used as the control. The final DNA extractions were amplified using primer pairs that cover regions flanking the p21waf1 transcriptional start site from −515 to +582 and then stained with ethidium bromide. The input was 1% of the soluble chromatin used for immunoprecipitation. (B) The 5′ genomic DNA sequence of human p21waf1 gene (AF497972). The messenger RNA coding sequence is in uppercase and the ZBP-89/Sp1/Sp3 binding element used for DAPA is boxed. The polymerase chain reaction primers for the ChIP assay are underlined. (C) HCT116 and U2OS cells were treated with 2.5 mmol/L sodium butyrate for up to 5 and 8 hours, respectively, the nuclear extracts were prepared and DAPA was performed using a biotin-labeled DNA element from −227 to −198 of the p21waf1 proximal promoter. The expression profiles and DNA binding patterns of ZBP-89, Sp1, and Lamin B were detected with their respective antibodies.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

4. Figure 3
HDACi stimulates formation of an ATM–ZBP-89 complex in vivo. (A) HCT116 cells expressing the Flag–ZBP-89 expression vector or the empty vector were treated with 2.5 mmol/L sodium butyrate for 16 hours. The nuclear extracts were prepared and the Flag–ZBP-89 protein complexes were immunoprecipitated with anti-Flag M2 beads and then eluted from the beads with FLAG peptide. The eluted proteins were separated on a sodium dodecyl sulfate–polyacrylamide electrophoresis gel and stained with Coomassie Brilliant Blue. MALDI-TOF mass spectrometry was used to identify the components in the resolved Flag–ZBP-89 immunoprecipitated complexes. (B) The immunoprecipitated Flag–ZBP-89 complexes were separated on a sodium dodecyl sulfate–polyacrylamide electrophoresis gel, transferred to membrane, and immunoblotted with anti-Flag or anti-ATM antibody. (C) HCT116 cells were treated with 2.5 mmol/L sodium butyrate or 1 μmol/L TSA for 1 hour. Nuclear extracts were prepared for immunoprecipitation with the rabbit anti–ZBP-89 antibody and ZBP-89 and ATM were detected with their respective antibodies in the immunoblotting analysis. (D) HCT116 cells were treated with 2.5 mmol/L sodium butyrate for up to 3 hours and soluble chromatin was prepared. ChIP assays were performed with goat anti-ATM IgG or goat IgG control. The final DNA extracted was amplified using primers that span from −246 to +46 of the p21waf1 promoter followed by ethidium bromide staining. The input was 0.375% of the soluble chromatin used for immunoprecipitation. (E) HCT116 cells were treated with 2.5 mmol/L sodium butyrate for 1 hour and nuclear extracts were prepared. The indicated proteins were immunodepleted with their respective polyclonal antibodies and the normal rabbit IgG was used as control. DAPA analyses were performed using a biotin-labeled DNA element spanning from −227 to −198 of the p21waf1 promoter.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

5. Figure 4
Reduction of ZBP-89 attenuated HDACi but not γ irradiation–induced ATMSer1981 and p53Ser15 phosphorylation. (A) HCT116 or A549 cells were transfected with a wild-type (WT) or mutant (Mut) siRNA oligonucleotide of ZBP-89 for 2 days, then treated with 2.5 mmol/L sodium butyrate or 1 μmol/L TSA for 8 hours before immunoblotting. (B) HCT116 cells were transfected with ZBP-89 siRNAs (ZBP) or control pGL2 luciferase siRNAs (Con) for 2 days, then the cells were irradiated at the indicated doses. After irradiation, the cells were allowed to recover for 30 minutes before harvesting for immunoblotting. (C) HCT116 cells were irradiated at 10 Gy, then were allowed to recover for the indicated time points before collection for immunoblotting analyses.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

6. Figure 5
HDACi induction of p21waf1 requires ATM. (A) GM00637I (normal fibroblast) or GM05849D (AT) cells were transfected with a p21waf1 luciferase reporter (0-Luc) along with pCMV-β-gal. The cells were treated with 2.5 mmol/L butyrate for 16 hours before collection for reporter assays. Shown is the mean of 3 independent experiments performed in triplicate ± standard error. *P < .05. (B) HCT116 or HCT116 p53 (−/−) cells were first transfected with control (scrambled sequences) or ATM siRNA for 1 day and then transfected with p21waf1 luciferase reporter (0-Luc) along with pCMV-β-gal. The cells were treated with 2.5 mmol/L butyrate for 16 hours before collection for reporter assays. Shown is the mean of 3 independent experiments performed in triplicate ± standard error. *P < .05; ***P < (C) HCT116 p53 (−/−) cells were transfected with scrambled sequences (Con), ZBP-89, ATM, or both ZBP-89 and ATM siRNAs for 2 days, then treated with 2.5 mmol/L sodium butyrate or 0.2 μmol/L TSA for 18 hours followed by immunoblot analyses.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

7. Figure 6
ATM associates with the zinc finger and amino terminal domains of ZBP-89. (A) HCT116 cells were transfected with deletion mutants of ZBP-89 for 2 days, then treated with 2.5 mmol/L sodium butyrate for 1 hour. The cells were harvested and immunoprecipitation was performed with anti-Flag antibody. (B) HCT116 cells were transfected with pcDNA3, pcDNA3-ZBP-89/FL, pcDNA3-ZBP-89/ΔZnF, or pcDNA3-ZBP-89/ΔNter expression vectors. Two days later, the cells were collected and nuclear extracts were prepared for DAPA using a biotin-labeled DNA element from −227 to −198 of the p21waf1 promoter. (C) HCT116 cells were transfected with pcDNA3, pcDNA3-ZBP-89/ΔZnF, or pcDNA3-ZBP-89/ΔNter expression vectors and were selected with G418 at 1 mg/mL for 2 weeks. The G418-resistant clones were pooled and treated with 2.5 mmol/L sodium butyrate for 16 hours before they were harvested for immunoblotting. Increased levels of exogenous ZBP-89 mutants (ΔZnF or ΔNter) were observed in the presence of butyrate because the cytomegaloviral promoter of pcDNA3 is GC-rich and butyrate inducible.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

8. Figure 7
The p300-interaction domain of ZBP-89 is essential for ATM phosphorylation in DSS-induced colitis. (A, B, E, F, I, and J) WT (ZBP-89FL/FL) or (C, D, G, H, K, and L) ZBP-89ΔN/ΔN mice were treated with 4% DSS for 5 days then returned to normal drinking water for 2 days. The colon sections were stained with (A, C, E, G, I, and K) H&E or (B, D, F, H, J, and L, green) fluorescein-conjugated phospho-ATMSer1981 antibody. 4′,6-diamidino-2-phenylindole was used for nuclei staining (blue). Shown are the representative sections from 3–5 mice in each group. MM, muscular mucosa. Magnification, 100×. (M) The colon sections from DSS-treated WT mice were double-stained for phospho-ATMSer1981 (green) and p21waf1 (red). 4′,6-diamidino-2-phenylindole was used for counterstaining of nuclei (blue). The arrow indicates the nuclear staining of p21waf1 (red) and the arrowheads indicate the colocalization of p21waf1 with phospho-ATMSer1981 (yellow). Magnification, 1000×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions