1. Volume 140, Issue 4, Pages 1261-1271.e1 (April 2011)
Intestinal DMT1 Cotransporter Is Down-regulated by Hepcidin via Proteasome Internalization and Degradation 
Carole Brasse–Lagnel, Zoubida Karim, Philippe Letteron, Soumeya Bekri, André Bado, Carole Beaumont 
Gastroenterology 
Volume 140, Issue 4, Pages e1 (April 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
Expression of DMT1 and FPN in Caco-2/TC7 cells. Caco-2/TC7 cells were grown for indicated times. DMT1 (A), ferroportin (FPN) (B), or sucrase isomaltase (SI) (C) messenger RNA (mRNA) levels were analyzed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and normalized to β-actin mRNA. Values are means ± standard error of mean for 3 independent experiments and are expressed as 2−ΔΔCt relative to that obtained with cells cultured for 3 days defined as 1. *P < .05 compared with cells cultured for 3 days, Student t test (n = 3). (D) Confocal analysis of DMT1 and FPN proteins transiently expressed as enhanced green fluorescence protein (EGFP)-fusion protein. Actin was stained using the phalloidine-Texas Red marker. Immunofluorescence images were obtained using a confocal microscopy system. (I) Focal plan. (II) Cross section from apical (top) to basolateral (bottom) side.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
Effect of hepcidin on 55Fe-transport by Caco-2/TC7 cells. (A) Caco-2/TC7 cells were incubated for 2 hours with different hepcidin concentrations (0, 100, 200, 500, and 1000 nmol/L) added at the basolateral side. For each condition, 55Fe-transepithelial transport was expressed as the ratio to control cells incubated without hepcidin. Data are mean ± standard error of mean (SEM) from triplicate samples of 3 independent experiments. *P < .05 compared with control medium defined as 1, n = 3, Student t test. (B) Time course of the changes in 55Fe-transepithelial transport induced by 200 nmol/L hepcidin. Data are mean ± SEM from triplicate samples of 3 independent experiments. *P < .05 compared with control medium at the indicated times, n = 3, Student t test. (C) 55Fe-associated radioactivity at the basolateral side of cells incubated for 2 hours without (control) or with 200 nmol/L hepcidin (Hep 200 2h), 100 ng/mL IL-6, or 1 nmol/L leptin or for 24 hours with hepcidin (Hep h). At the indicated time, data are expressed as the ratio to cells incubated without hepcidin and are the mean ± SEM from triplicate samples of 3 independent experiments. *P < .05 compared with control medium at the indicated times, n = 3, Student t test. (D) Cells were treated with or without (C) 200 nmol/L hepcidin or 100 ng/mL IL-6 for 2 hours. The inserts were then challenged for 30 minutes with 55FeNTA, and the 55Fe content of cells was quantified. Data are means ± SEM from triplicate samples of 3 independent experiments. *P < .05 compared with control medium defined as 1, n = 3, Student t test.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Effect of hepcidin on DMT1 and FPN protein amount in Caco-2/TC7. (A) Western blotting of cells incubated for 2 hours with 200 nmol/L hepcidin, showing DMT1 (≈100 kilodaltons), FPN (≈70 kilodaltons), and β-actin (≈42 kilodaltons). Molecular weight markers are indicated on the left. (B) Densitometric analysis of the bands. Values are means ± standard error of mean (SEM) for 5 independent experiments and expressed as the ratio DMT1 (solid bars) or FPN (open bars)/β-actin ratio normalized to 1.0 for untreated cells. *P < .05 compared with control, n = 5, Student t test. (C) Immunofluorescence imaging of live Caco-2/TC7 cells transfected with a (+IRE) DMT1- or FPN-enhanced green fluorescence protein (EGFP) encoding vector. Three days after transfection, cells were incubated with (hepcidin) or without (vehicle) 200 nmol/L hepcidin. Images were obtained before (0h) or 2 hours (2h) after hepcidin addition. (D) Western blot analysis of J774 cells incubated with 10 μmol/L ferric ammonium citrate (FAC) for 24 hours with 200 or 700 nmol/L hepcidin for 2 hours. Molecular weight markers are indicated on the left. (E) DMT1 messenger RNA (mRNA) level was normalized to β-actin mRNA. Values are means ± SEM for 3 independent experiments and are expressed as 2-ΔΔCT relative to that obtained with untreated cells defined as 1. *P < .05 compared with untreated cells, Student t test, n = 3.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Fig. 4
Influence of 200 nmol/L hepcidin on ex vivo duodenal segments of mice. (A) Apical to basolateral 55Fe-transport from isolated duodenal segments expressed as a ratio to the value at time 2 minutes. *P < .05 vs control mice at the indicated time, n = 5, Mann-Whitney test. (B) Western blotting of crude membrane extracts from 4 different duodenal loops for each condition after 30-minute incubation with or without hepcidin 200 nmol/L, showing DMT1 (≈100 kilodaltons), FPN (≈70 kilodaltons), and β-actin (≈42 kilodaltons). Molecular weight markers are indicated on the left. (C) Densitometric analysis. Values are expressed as median ± standard error of mean of the DMT1 (shaded box) or FPN (open box)/β-actin ratio. *P < .05 compared with duodenal segment without hepcidin, n = 4, Mann-Whitney test.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
Involvement of proteasome in the hepcidin-mediated decrease in DMT1. (A) Western blotting of Caco-2/TC7 cells incubated for 1 hour with 50 μmol/L MG-132 and then with or without 200 nmol/L hepcidin for 1 or 2 hours, showing DMT1, FPN, and β-actin. (B) Densitometric analysis. Values are means ± standard error of mean (SEM) for 4 independent experiments and expressed as the DMT1/β-actin ratio normalized to 1 for untreated cells. *P < .05 compared with controls. Student t test. (C) Three days after transfection, (+IRE) DMT1-enhanced green fluorescence protein (EGFP) transfected cells were incubated for 2 hours with or without 200 nmol/L hepcidin. Cell extracts were immunoprecipitated with EGFP antibody, and the immunoprecipitates were analyzed by Western blotting using the antiubiquitin (Ubq) or anti-EGFP antibodies as indicated. Representative autoradiograms of 3 independent experiments are shown. (D) Densitometric analysis. Values are expressed as median ± SEM of ubiquitin/DMT1 ratio. *P < .05 compared with t = 0, n = 4, Student t test.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Figure 6
Involvement of ubiquitination on iron transport and hepcidin effect. (A) Caco-2/TC7 treated for 1 hour without or with MG-132 (50 μmol/L) or PYR-41 (50 μmol/L) were then incubated for 2 hours with (open bars) or without (solid bars) 200 nmol/L hepcidin added to the basolateral side. 55Fe-transepithelial transport was expressed as the ratio to cells incubated without hepcidin and without inhibitor (control). Data are mean ± standard error of mean (SEM) from triplicate samples of 3 independent experiments. *P < .05 compared with control medium, n = 3, Student t test. (B) Three days after transfection, FPN-eGFP transfected cells were incubated with or without 200 nmol/L hepcidin. Protein immunoprecipitated with EGFP antibody were subjected to immunoblotting by using the antibodies against phosphotyrosin (PTyr) or FPN, respectively. A representative autoradiogram of 3 independent experiments is shown, with detection of the native EGFP-FPN protein or its phosphorylated form. NT, immunoprecipitated proteins from nontransfected cells; PE, protein extract from non transfected cells without immunoprecipitation; IP, immunoprecipitation.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions