1. Volume 127, Issue 4, Pages 1139-1149 (October 2004)
Apobec-1 protects intestine from radiation injury through posttranscriptional regulation of cyclooxygenase-2 expression 
Shrikant Anant, Nabendu Murmu, Courtney W. Houchen, Debnath Mukhopadhyay, Terrence E. Riehl, Stephen G. Young, Aubrey R. Morrison, William F. Stenson, Nicholas O. Davidson 
Gastroenterology 
Volume 127, Issue 4, Pages (October 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Stem cell survival in APOBEC-1−/− mice is reduced after radiation injury. (A) Experimental protocol. WT, apoB100-only, and APOBEC-1−/− mice were treated with 12 Gy of γ-irradiation; 82 hours after radiation, BrdU was administered, and the mice were killed 2 hours later. (B) The distal jejunum was stained with H&E for morphological analysis. To identify dividing cells, the tissue was immunostained for BrdU and counterstained with eosin. (C) Total number of regenerating crypts per cross section; values are mean ± SEM (n = 5 mice per genotype). (D) Number of BrdU-positive cells. Values are mean ± SEM (n = 5 mice per genotype). (E) Total number of regenerating crypts per cross section/time course. Values are mean ± SEM (n = 4 mice per genotype). (F) Total number of regenerating crypts/dose curve. Values are mean ± SEM (n = 4 mice per genotype). (G) Epithelial cells at positions 3 to 8 from the base of the crypts are preferentially protected from apoptosis in WT mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 1
Stem cell survival in APOBEC-1−/− mice is reduced after radiation injury. (A) Experimental protocol. WT, apoB100-only, and APOBEC-1−/− mice were treated with 12 Gy of γ-irradiation; 82 hours after radiation, BrdU was administered, and the mice were killed 2 hours later. (B) The distal jejunum was stained with H&E for morphological analysis. To identify dividing cells, the tissue was immunostained for BrdU and counterstained with eosin. (C) Total number of regenerating crypts per cross section; values are mean ± SEM (n = 5 mice per genotype). (D) Number of BrdU-positive cells. Values are mean ± SEM (n = 5 mice per genotype). (E) Total number of regenerating crypts per cross section/time course. Values are mean ± SEM (n = 4 mice per genotype). (F) Total number of regenerating crypts/dose curve. Values are mean ± SEM (n = 4 mice per genotype). (G) Epithelial cells at positions 3 to 8 from the base of the crypts are preferentially protected from apoptosis in WT mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 2
LPSprotects WT but not APOBEC-1−/− mice from radiation-induced apoptosis. (A) Western blot analyses for COX-2 and HSP40 proteins of lysates from the distal jejunum of mice subjected to 12 Gy of γ-irradiation. The mobility of the molecular weight markers is shown on the left. (B) Schematic representation of treatment regimen. Mice were injected with LPS (10 mg/kg) or dimethyl PGE2 (0.5 mg/kg) 14 hours before γ-irradiation (12 Gy). The mice were killed 6 hours later, and distal jejunum was collected for assessment of apoptosis and PGE2 levels. (C) Radiation-induced apoptosis was assessed by counting the total number of apoptotic cells per crypt in WT and APOBEC-1−/− mice. Values are mean ± SEM (n = 5 mice per genotype). (D) Intestinal PGE2, expressed as mean ± SEM (n = 5 mice per genotype); P <0.001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 3
COX-2 is not induced in APOBEC-1−/− mice. (A) Mice were treated with LPS 14 hours before irradiation and were killed 6 hours later. Mice treated with LPS only were killed at the time of irradiation. (B) Real-time polymerase chain reaction (PCR) was performed with total RNA from the distal jejunum and expressed as the amount of COX-2 mRNA relative to the glyceraldehyde phosphate dehydrogenase control for each genotype. (C) Lysates of distal jejunum were subjected to Western blot analyses for COX-2, apobec-1, and HSP40 proteins. The mobility of the molecular weight markers is shown on the left. (D) Real-time PCR for c-myc mRNA was performed with total RNA from the distal jejunum and quantitated relative to the glyceraldehyde phosphate dehydrogenase control for each genotype. aP = (E) Real-time PCR for c-myc mRNA was performed from distal jejunal RNA from mice subjected to 12 Gy of γ-irradiation. aP < compared with WT mice at time 0; bP = compared with WT mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 4
Apobec-1 interacts with AU-rich sequences in the COX-2 3′ UTR. (A) Electrophoretic mobility shift assay was performed with recombinant glutathione S-transferase (GST)/APOBEC-1 and radiolabeled templates. Probes used in the assay were RB (105 nt of rat apoB RNA containing nt 6639–6743), vascular endothelial growth factor (VEGF; 1.9 kilobases [kb] of 3′ UTR of human VEGF mRNA), COX (2.3 kb of 3′ UTR of mouse COX-2 mRNA), and IL-8 (1.3 kb of 3′ UTR of human IL-8 mRNA). Locations of the apobec-1 bound (b) and free (f) probe are indicated to the right. (B) Apobec-1-bound RNA, isolated from intestinal epithelial cells of WT and APOBEC-1−/− mice exposed to 12 Gy of whole-body γ-irradiation, was subjected to reverse-transcription polymerase chain reaction for COX-2 and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNAs. T, total RNA; P, pellet; S, supernatant. (C) Schematic representation of substitutional mutants generated from the first 60 nt of the COX-2 3′ UTR. Three sets of AU-rich sequences (A-I-A-III) are present in this region. Sequences underlined in each mutant were substituted with the complementary sequences. (D) UV crosslink analysis was performed with recombinant GST/APOBEC-1 and radiolabeled WT and mutant COX-2 3′ UTR transcripts. The arrow indicates the location of the crosslinked band. The mobility of the molecular weight markers is shown on the left.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 5
Apobec-1 stabilizes luciferase (Luc) mRNA containing the first 60 nt of the COX-2 3′ UTR. (A) Schematic representation of Luc plasmids containing the various 3′ UTR regions of the COX-2 mRNA. **AREs within the first 60 nt of COX-2 3′ UTR. (B) The Luc plasmids containing the various COX-2 3′ UTRs were co-transfected with either apobec-1-expressing or β-galactosidase (LacZ)-expressing plasmids into HEK293 cells. Actinomycin D (10 μg/mL) was added to the medium 48 hours after transfection, and the cells were incubated for the indicated times. Total RNA was extracted and subjected to Northern blot analysis for Luc and glyceraldehyde phosphate dehydrogenase mRNAs. Shown is a representative figure of 3 such experiments. (C) The half-life of the Luc mRNAs containing various COX-2 3′ UTRs; values are mean ± SEM of 3 transfections. FL, full length.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 6
The p38 MAPK inhibitor SB (SB) does not affect apobec-1-mediated COX-2 mRNA stability. (A) HeLa cells were infected with recombinant adenovirus constructs carrying β-galactosidase or apobec-1 for 16 hours. SB was added to a final concentration of 20 μmol/L, cells were incubated for a further 6 hours, and lysates were subjected to Western blot analyses for apobec-1 and HSP40. The mobility of the molecular weight markers is shown on the left. (B) Total RNA was isolated at various times after the addition of actinomycin D and subjected to real-time polymerase chain reaction analyses for COX-2 mRNA. The data are presented as COX-2 mRNA levels present relative to those observed at the addition of actinomycin D.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions