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SUPPLEMENTAL MATERIAL AND METHODS
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Infection of AGS cells with Helicobacter species H. pylori B128 and SS1 were
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grown on 10% blood agar plates under microaerophilic conditions. Bacteria were
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added to AGS cells at a Multiplicity Of Infection (MOI) of 200 or 600 per cell for 24
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hours. After cells were harvested, total RNA was extracted from cells using Trizol
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(Life technologies) according to the instructions provided by the manufacturer.
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Reverse transcription was done with 2 to 3 μg of total RNA as described earlier.[1]
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Quantitative PCR was performed with 2 μl of a 1/10 dilution of reverse-transcribed
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total RNA dilution, 10 μM of each primer and 2 mM MgCl2 in 1x LightCycler DNA
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Master SYBR Green I mix using a LightCycler apparatus (Roche Applied Science).
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All samples were normalized to the threshold cycle value for 18S.
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In situ hybridization Cellular localization of hepcidin mRNA in the stomach was
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analyzed using in situ hybridization following a method described earlier with
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modifications.[2] Mouse hepcidin-specific sense and antisense DNA probes were
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PCR
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ctaatacgactcactataggg-CAGCAGAACAGAAGGCATGA-3’ (forward sense primer for
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antisense probe), 5’-GGGGAGGGCAGGAATAAATA-3’ (reverse sense primer for
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antisense
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(forward antisense primer for sense probe) and 5’-CAGCAGAACAGAAGGCATGA-3’
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(reverse antisense primer for sense probe; small letters indicate the T7 promotor
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sequence). Briefly, DNA probes were reversely transcribed into RNA probes using T7
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RNA polymerase and digoxigenin-labelled (Roche Diagnostics, Indianapolis, USA).
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Resulting antisense and sense probes (each 200 µg ml-1) were hybridized to
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deparaffinized and dehydrated tissue sections preincubated with 1% hydrogen
amplified
from
probe),
HepG2
cDNA
using
the
following
primers:
5’-
5’-ctaatacgactcactataggg-GGGGAGGGCAGGAATAAATA-3’
1
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peroxidase and pre-digested with proteinase K. After blocking endogenous biotin
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(Dako Germany, Hamburg) and amplify the signal with an anti-DIG antibody
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conjugated with alkaline phosphatase (Roche, Mannheim, Germany) signal was
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visualized by incubating sections with NBT and BCIP (Roche). The resulting blue
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staining was examined on a light microscope (Leica, Solms, Germany) and images
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were acquired with a digital camera connected to the microscope and Leica
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Application Suite software (Leica Microsystems, Heerbrugg, Switzerland).
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Isolation of rat parietal cells All reagents (of analytical grade) were obtained from
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Sigma (Taufkirchen, Germany) or Merck (Darmstadt, Germany).
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Gastric epithelial cells were isolated as previously described.[3] Two male nonfasted
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Sprague Dawley rats (250 g) were sacrificed by cervical dislocation. The stomachs
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were excised, everted to form sacs and rinsed with 0.9% saline. Three ml of Pronase
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E solution (1.4 mg/ml buffer A [0.5 mM NaH2PO4, 1 mM Na2HPO4, 20 mM NaHCO3,
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70 mM NaCl, 5 mM KCl, 11 mM glucose, 50 mM Hepes, 0.8 mM Na2-EDTA, and 10
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mg ml-1 BSA, pH 7.8]) were injected into the lumen of each sac. The stomachs were
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then incubated for 35 min at 37°C in oxygenated buffer A. Superficial mucosal cells
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were discarded after incubating the stomachs for 10 min in oxygenated buffer B [as
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buffer A but without Na2-EDTA and with 1.0 mM CaCl2, 1.5 mM MgCl2, and 5 mg ml-1
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BSA]. After another 30 min incubation in oxygenated buffer A, mucosal cells were
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dissociated by 8 min of stirring in 60 ml buffer B and collected by centrifugation at
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180xg for 3 min after filling up to 100 ml with buffer C [140 mM NaCl, 1.2 mM MgSO 4,
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1.0 mM CaCl2, 10 mM Hepes; 11 mM glucose, 0.6 mM dithiothreitol, and 0.1% (w/v)
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BSA, pH 7.4]. Subsequently, the stomachs were incubated in buffer A for another 20
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min, followed by two further collection steps in buffer B (for 8 and 10 min,
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respectively). The collected cells were resuspended and washed in 50 ml buffer C
2
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and centrifuged at 180xg for 3 min. After a second wash in buffer C, cells were
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pooled and resuspended in 10 ml buffer C.
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For enrichment of parietal cells, the crude cell suspension was separated according
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to increasing cell diameters by counterflow elutriation (rotor JE-6B; equipped with
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standard chamber; centrifuge J2-21, Beckman instruments, München, Germany) as
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described.[4] Briefly, the crude suspension of isolated cells was loaded into the rotor
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at a flow rate of 16 ml/min and a rotor velocity of 2,400 rpm. Erythrocytes, cell
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fragments and cells with diameter <14 µm were removed by washing at 40 ml/min
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and 2,000 rpm. Thereafter, a 300 ml cell fraction was collected at 62 ml/min and
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2,000 rpm. This fraction consisted of about 80-85% parietal cells. The viability of
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enriched parietal cells was determined by trypan blue exclusion and exceeds mostly
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95%. The enriched parietal cells were centrifuged at 180xg for 3 min and used for
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RNA isolation as described in Materials and Methods.
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3
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SUPPLEMENTAL FIGURE LEGENDS
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Figure S1. TNFα and H+/K+-ATPase in H. plyori infected patients.
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A: Expression levels of tumor necrosis factor (TNFα) in corpus and antrum biopsiens
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obtained from H. pylori infected patients (n=13) and uninfected control patients
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(n=22) were determined by quantitative RT-PCR.
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Expression level H. pylori negative controls was defined as one and results are
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presented as means ± SD of relative changes (presented as TNFalpha/ßactin ratio).
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Figure S2. Histochemical examination of WT and hepcidin KO mice stomach
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tissue. Representative photographs of H&E (A) and Periodic acid-Schiff staining with
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hemotoxylin counterstaining (B) in fundus/corpus and antrum tissue sections of WT
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and KO mice (aged 6 month). Scale bars (50 µm) in the lower left pictures are
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representative for all pictures.
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Figure S3. Marker of inflammation in hepcidin KO mice.
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Determination of IL-6 (A), interferon γ (IFNγ; B), and tumor necrosis factor alpha
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(TNFα; C) expression levels by quantitative RT-PCR in stomach of hepcidin KO mice
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and age and sex-matched WT mice (black bars: 4 month old mice, white bars: 6
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month old mice). Expression level in WT was defined as one and results are
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presented as means ± SD of relative changes. *p<0.05
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Figure S4. Hepcidin expression is affected by inhibition and activation of
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gastric acid secretion.
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A+B: Hepcidin/GAPDH mRNA ratio analyzed in PPI-treated wildtype mice (A; n=10
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per group) or in mice subjected to histamine injection (B; n=5 per group). Expression
4
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level in NaCl-injected animals were set to one and relative changes in expression
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levels (presented as hepcidin/GAPDH ratio) in PPI or histamine-injected mice were
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shown as mean ± SD. *p<0.05, **p<0.005.
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5
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SUPPLEMENTAL TABLES
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Table S1: Quantitative RT-PCR primers
species
gene
forward primer
reverse primer
mouse
hepcidin1 (Hamp1)
5’-aacagataccacactgggaa-3’
5’-cctatctccatcaacagatg-3’
H+/K+-ATPase (Atp4a)
5’-catgactgccaagatgagc-3’
5’-cgggcaaacttcacatactc-3’
gastrin receptor (Cckbr)
5´-ccagagaattggagttgacc-3´
5´-agggagagtaggaaggcgtt-3´
gastrin (Gast)
5’-gctggctctagctaccttct-3’
5’-gacaggtctgctatgaagtg-3’
somatostatin receptor (Sst2r)
5´-gttcatctacttggtggtgt-3´
5´-ttcatctgcaatggccaggt-3´
somatostatin (Sst)
5´-ggaaacaggaactggccaag-3´
5´-gccattgctgggttcgagtt-3´
Interleukin 6 (Il6)
5'-tcggaggcttaattacacatgttct-3'
5'-gcatcatcgttgttcatacaatca-3'
gastric intrinsic factor (Gif)
5’-cttcaatgtgaacacaggag-3’
5´-tgctgtagatgtctccgatg-3´
TNFalpha (Tnf)
5’-tcagcctcttctcattcctgctt-'3
5’-aggccatttgggaacttctcatc-'3
IFNgamma (Ifng)
5’-gaggtcaacaacccacaggt-'3
5’-gactccttttccgcttcctg-'3
ß-actin (Actb)
5'-ccaaaagccacccccactc-'3
5'-ggggacaaaaaaaagggagg-3'
GAPDH (gapdh)
5'-catgaccacagtccatgcca-'3
5'-tcagatccacgacggacaca-3'
hepcidin (Hamp)
5’-ggcagaaagcaagactgatgac-3’
5’-acaggaataaataatggggcg-3’
H+/K+-ATPase (Atp4a)
5’-gagaagaagtcatgcagccag-3’
5’-caatggtctgcatcagcac-3’
ß-actin (Actb)
5’-tgacgttgacatccgtaaagac-3’
5’-cagtgaggccaggatagagc-3’
hepcidin (HAMP)
5’-ctgcaaccccaggacagag-3’
5’-ggaataaataaggaagggagggg-3’
Interleukin 6 (IL6)
5’-cagccctgagaaaggagaca-3’
5’-ccaggcaagtctcctcattg-3’
H+/K+-ATPase (ATP4a)
5'-ctgtactacgtggccttctac-3'
5'-gtgcagttgatgctgtcctc-3'
TNFalpha (TNF)
5’-ccaggcagtcagatcatctt-3’
5’-cagctggttatctctcagct-3’
ß-actin (ACTB)
5’-aggatgcagaaggagatcactg-3’
5’-gggtgtaacgcaactaagtcatag-3’
rat
human
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Table S2: Characteristics of patients with and without H. pylori infection
State at diagnosis
H. pylori positive
H. pylori negative
13
22
10/3
11/11
23-65 (44)
22-65 (42)
patients
males/females
age range (median)
H. pylori positive
H. pylori negative
-
3
6
-
serum iron [µmol l-1]
13.6-36.1 (17.1)
8.1-26.5 (13.3)
9.1-30.5 (17.1)
serum ferritin [ µg l-1]
82.0-343.0 (137.0)
17.0-153.0 (94.5)
10.0-364.0 (141.5)
1.8-2.4 (2.1)
1.9-2.8 (2.1)
1.5-2.9 (2.1)
24.3-81.2 (32.8)
11.6-54.9 (24.1)
15.5-78.3 (28.6)
State after eradication
patients (*)
serum transferring [g l-1]
serum transferrin saturation [%]
103
* 4 patients refused to participate in the study after the treatment
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SUPPLEMENTAL REFERENCES
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1.
Nicolas G, Chauvet C, Viatte L et al. The gene encoding the iron regulatory
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peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin
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Invest 2002;110: 1037–1044.
109
2.
Zhong B, Strnad P, Toivola DM et al. Reg-II is an exocrine pancreas injury-
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response product that is up-regulated by keratin absence or mutation. Mol Biol
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Cell 2007;18:4969-4978.
112
3.
of IL-1 receptors on rat parietal cells. Am J Physiol 1998;275:G1094-G1105.
113
114
Schepp W, Dehne K, Herrmuth H et al. Identification and functional importance
4.
Schmidtler J, Schepp W, Janczewska I et al. Glucagon-like peptide-1 (GLP-1):
115
potent cAMP-dependent stimulus of rat parietal cell H+ production. Am J Physiol
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1991;260:G940-G950.
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