1. Volume 126, Issue 4, Pages 1030-1043 (April 2004)
Role of interferon-stimulated responsive element-like element in interleukin-8 promoter in Helicobacter pylori infection 
Yoshio Yamaoka, Takahiko Kudo, Hong Lu, Antonella Casola, Allan R. Brasier, David Y. Graham 
Gastroenterology 
Volume 126, Issue 4, Pages (April 2004)
DOI: /j.gastro

2. Figure 1
Adherence ability of the H. pylori strains to MKN45 cells. WT H. pylori JK51 and its oipA, hopZ, oipA/hopZ, or cagE KO mutants were used. The data and error bars represent the mean and SE. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < compared with WT strain.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Time course kinetics of IL-8 protein production from MKN45 cells cocultured with H. pylori JK51. The data and error bars represent the mean and SE. •: WT strain, ▴: hopZ KO mutant, ○: oipA KO mutant, ■: cagE KO mutant, □: cagE/oipA double KO mutant.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
IL-8 promoter activation following H. pylori infection (A and B) or TNF-α stimulation (A). MKN45 cells were transiently transfected with LUC reporter plasmid containing a −162/+44 fragment of the hIL-8 promoter and infected with WT strain JK51 and its hopZ or oipA KO mutants (A), cagE or cagE/oipA KO mutants (B), or stimulated with TNF-α (10 ng/mL). Untreated plates served as controls. For each plate, luciferase activity was normalized to renilla luciferase vector DNA. Data are expressed as mean ± SE of normalized luciferase activity. Numbers on the error bars refer to fold increase of luciferase activity in H. pylori-infected or TNF-α-stimulated cells relative to untreated controls.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Schematic representation of human IL-8 promoter/luciferase reporter plasmid (A), effects of 5′ deletions in the IL-8 promoter sequence on H. pylori and TNF-α-inducible activity (B), and effect of 5′ deletions in the IL-8 promoter sequence on H. pylori-inducible activity (C). Locations of binding sites for ISRE-like element, AP-1, NF-IL6, and NF-κB are illustrated in A. (B) MKN45 cells were transiently transfected with 5′ deletions of the hIL-8 LUC promoter and either infected with WT H. pylori JK51 or stimulated with TNF-α (10 ng/mL) for 6 hours. Untreated plates served as controls. For each plate, luciferase activity was normalized to renilla luciferase vector DNA. Data are expressed as mean ± SE of normalized luciferase activity. Numbers on the error bars refer to fold increase of luciferase activity in H. pylori-infected or TNF-α-stimulated cells relative to untreated controls. (C) MKN45 cells were transiently transfected with 5′ deletions of the hIL-8 LUC promoter and infected with WT H. pylori JK51 or oipA KO mutants for 6 hours. Shown is the normalized luciferase activity expressed as fold induction. Data are expressed as mean ± SE of normalized luciferase activity. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < from −162/+44; ##P < 0.01, ###P < from −132/+44; ††P < 0.01, †††P < from −99/+44 among the same stimulus.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Effect of site mutations in the IL-8 promoter sequence on H. pylori and TNF-α-inducible activity. (A) MKN45 cells were transiently transfected with site-mutated plasmids of the −162 IL-8 gene promoter and either infected with WT H. pylori JK51 or stimulated with TNF-α (10 ng/mL) for 6 hours. Untreated plates served as controls. For each plate, luciferase activity was normalized to renilla luciferase vector DNA. Data are expressed as mean ± SE of normalized luciferase activity. Numbers on the error bars refer to fold increase of luciferase activity in H. pylori-infected or TNF-α-stimulated cells relative to untreated controls. (B) MKN45 cells were transiently transfected with site-mutated plasmids of the −162 IL-8 gene promoter and infected with WT H. pylori JK51 or oipA knockout mutants for 6 hours. Shown is the normalized luciferase activity expressed as fold induction. Data are expressed as mean ± SE of normalized luciferase activity. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < from −162/+44 among the same stimulus.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
ISRE-like element can partly restore the WT H. pylori inducibility in the −99 IL-8 promoter. (A) Schematic representation of the 2 (ISRE-like elements) −99/+44 IL-8 LUC plasmid. (B) MKN45 cells were transiently transfected with the different IL-8 promoter constructs and infected with WT H. pylori JK51 or oipA KO mutants for 6 hours. Shown is the normalized luciferase activity expressed as fold induction. Data are expressed as mean ± SE of normalized luciferase activity.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
EMSA of IL-8 ISRE-like element binding complexes (A and B), IL-8 TNFRE binding complexes (C), and IL-8 AP-1 binding complexes (D) in response to H. pylori infection: Nuclear extracts were prepared from control and H. pylori-infected MKN45 cells (3 hours) and used for EMSA. In competition analysis, the presence of super shifted band is indicated by arrow. (B) Lanes 1, 2, and 3: Competition analysis. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used to bind to ISRE-like element prove; 5 pmol/L of unlabeled competitor (WT prove [WT] or mutated prove [Mut]) was added in the binding reaction. Lanes 4 and 5: Super shift assay. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used in the EMSA in the presence of anti-IRF-1, -IRF-2, and -IRF-3 antibodies. (C) Lanes 5 and 6: Competition analysis. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used to bind to TNFRE prove; 5 pmol/L of unlabeled competitor (mutated in the NF-IL6 site [ΔIL] or in the NK-κB site [ΔK]) were added in the binding reaction. Lanes 7 and 8: Super shift assay. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used in the EMSA in the presence of anti-p50 and -p65 antibodies. (D) Lanes 5 and 6: Competition analysis. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used to bind to AP-1 prove; 5 pmol/L of unlabeled competitor (AP-1 or NF-IL6) were added in the binding reaction. Lanes 7 and 8: Super shift assay. Nuclear extracts of MKN45 cells infected with WT strain JK51 for 3 hours were used in the EMSA in the presence of anti-c-Fos and -c-Jun antibodies.
Gastroenterology  , DOI: ( /j.gastro )

9. Figure 8
Western blot analysis for nuclear IRF-1 protein induction. WT H. pylori JK51 was cocultured with MKN45 cells for 3 hours, and nuclear protein was extracted and used for Western blot.
Gastroenterology  , DOI: ( /j.gastro )

10. Figure 9
Western blot analysis for STAT1, IκBα, JNK, and ERK antibody (phospho-specific [upper] and control antibody): WT H. pylori JK51 was cocultured with MKN45 cells for 120 minutes, and total protein was extracted and used for Western blot.
Gastroenterology  , DOI: ( /j.gastro )

11. Figure 10
Phosphorylated STAT1 levels in the antral gastric mucosa. The end of the bars indicates the 25th and 75th percentiles. The median is indicated with a solid line in the box, and the 10th and 90th percentiles are indicated with error bars. Phosphorylated STAT1 levels were adjusted by total STAT1 levels (units/pg total STAT1 levels).
Gastroenterology  , DOI: ( /j.gastro )