1. Volume 131, Issue 2, Pages 497-509 (August 2006)
The Role of Tumor Necrosis Factor α in Down-Regulation of Osteoblast Phex Gene Expression in Experimental Murine Colitis 
Jennifer K. Uno, Olga I. Kolek, Eric R. Hines, Hua Xu, Barbara N. Timmermann, Pawel R. Kiela, Fayez K. Ghishan 
Gastroenterology 
Volume 131, Issue 2, Pages (August 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
(A) Real-time PCR analysis of colonic TNF-α mRNA expression in chemically induced colitis in mice fed control or curcumin-supplemented diets. Values are means ± SE; n = 3. *P < .05 as indicated by Fisher’s PLSD. (B) The effect of curcumin on LPS-stimulated (10 ng/mL) TNF-α mRNA expression in YAMC (white bars, left axis) and RAW cells (grey bars, right axis). Values are means ± SE; n = 4. *P < .05 between groups for respective cell lines. (C) Quantification of Phex mRNA expression in calvaria of mice with chemically induced colitis fed control or curcumin-supplemented diets. Values are means ± S.E.; n = 3–4. Data from all panels were analyzed by ANOVA followed by Fisher’s PLSD post-hoc test. *P < .05 between Control or TNBS+Curcumin groups and TNBS.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
Exogenous TNF-α administration inhibits Phex mRNA expression in mouse calvaria. Phex mRNA expression was analyzed by real-time PCR in bone harvested from C57BL/6 mice 24 hours after administration of PBS (control) or TNF-α (IP, 15 μg/g BW; TNF-α). Values are means ± SE of 3 independent experiments, * P = .004 as indicated by Student t test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Neutralizing anti-TNFα antibody restores Phex mRNA expression in TNBS-treated animals. Phex mRNA expression was analyzed by real-time PCR in bone harvested from control Balb/c mice or mice treated with TNBS or TNBS + Anti–TNF-α. Anti–TNF-α antibodies were given to mice (250 μg/mouse) 4 hours prior to TNBS enema and again on day 3 after TNBS treatment. Values are means and SE of 6–8 independent experiments. *P = for Control vs. TNBS, #P < for TNBS vs. Anti–TNF-α, # P = for Control vs. Anti–TNF-α; ANOVA followed by Fisher’s PLSD post-hoc test).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Endogenous Phex mRNA expression in UMR-106 osteoblasts is inhibited by TNF-α in a concentration-dependent fashion. Endogenous Phex mRNA expression was evaluated by real-time PCR in UMR-106 cells treated with PBS or increasing concentrations of TNF-α (24-hour treatment). Values are means ± SE of 3 independent experiments. Data were analyzed by ANOVA followed by Fisher’s PLSD post-hoc test. *P < .05 between control (PBS) and respective TNF-α concentration.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Inhibition of UMR-106 osteoblastic mineralization by TNF-α correlates with decreased expression of membrane-associated Phex protein. (A) Mineralization is expressed as fluorescence of calcium phosphate deposition (calcein staining) in UMR-106 cells treated with PBS or TNF-α (10 ng/mL) for 24, 48, or 72 hours. Values are means ± SE of 3 independent experiments; *P < .05 and **P < .01 as indicated by Student t test. (B) A representative confocal imaging of calcein staining following 72 hours of treatment. (C) Immunoblot analysis of Phex gene protein expression in hyp and normal animals (left panel) demonstrating specificity of the raised antiserum, and in PBS or TNF-α–treated UMR-106 cells following 72 hours of treatment under mineralization conditions (middle panel). Summary of densitometric results are represented in the right panel, *P = (Student t test; n = 5).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Phex mRNA stability is not affected by TNF-α treatment in UMR-106 osteoblasts. UMR-106 cells were treated with actinomycin D to inhibit active transcription, and Phex mRNA decay was studied by real-time PCR analysis of PBS-treated (filled circles) or TNF-α–treated cells (10 ng/mL; open circles). No significant differences in Phex transcript decay were detected. Values are means ± SE of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
TNF-α treatment decreases Phex gene promoter activity in a concentration- and time-dependent fashion in transiently transfected UMR-106 cells. (A) Phex promoter activity, expressed as β-galactosidase activity (relative light units per microgram of protein) calculated as fold change over PBS-treated cells. Transfected UMR-106 cells treated with increasing concentrations of TNF-α for 24 hours. Values are means ± SE of 4 independent experiments (*P < .05 between control [PBS] and respective TNF-α concentration; ANOVA and Fisher’s PLSD post-hoc test). (B) Phex promoter activity, expressed as Renilla luciferase activity calculated as fold over PBS-treated cells at a designated time point. Transfected UMR-106 cells were treated with 10 ng/mL TNF-α for 30 minutes to 24 hours. Values are means ± SE of 4 independent experiments. Asterisks indicate a statistically significant change (*P < .05; t test) at 16 and 24 hour time points. For both panels, data were analyzed by ANOVA followed by Fisher’s PLSD post-hoc test. *P < .05 for changes significantly different from the .5, 2.0, and 8.0 hour time points.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

9. Figure 8
−133 to −74 nt region of the Phex promoter is likely involved in the mechanism of TNF-α–mediated transcriptional repression. UMR-106 cells were transiently transfected with progressive deletion constructs of murine Phex promoter and treated with PBS (white bars) or TNF-α (10 ng/mL, 24 hours; black bars). Promoter activity is expressed as Renilla luciferase activity (relative light units per microgram of protein). Values are means ± SE of 3–4 independent experiments; *P < .05 for PBS versus TNF-α treatment as indicated by Student t test. pGL4.72 Basic represents promoterless reporter vector.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions