table s1. primer sequences. shown are sequences of - blood
TRANSCRIPT
Table S1. Primer sequences. Shown are sequences of primer sets used for the expression analysis of iron metabolism genes by real-
time PCR.
Gene Accn Gene Name Forward Primer Reverse Primer
Cp NM_001042611 Ceruloplasmin AGG AGT ATG AGG GAG CCG TCT A TTT GTC ATC AGC CCG TTG AA
Heph NM_010417 Hephaestin GCT CTG GCT CTT GGT GGT GTA TGT TGC GCC GAA GCT TTC
Aco1 NM_007386 Aconitase 1 CCT GTG TGC TCC GGG AAC T GCT TAT TGA TGG TCA CGT GTC TCT
Ireb2 NM_022655 Iron responsive element binding protein 2 GCT ATG AGG GAG GCA GTG AAA A GGA CAG GCA GGG TGG ACT TT
Hamp NM_032541 Hepcidin antimicrobial peptide TGT CTC CTG CTT CTC CTC CT CTC TGT AGT CTG TCT CAT CTG TTG
Hfe NM_010424 Hemochromatosis TGA TCT GCA GCC TGC TGA AC TGC CCC CTC CAA GTC TTT G
Hfe2 NM_027126 Hemochromatosis type 2 ATC CCC ATG TGC GCA GTT T CTC CTT GGA CAC GGC ATG T
Mfrn NM_026331 Solute carrier family 25, member 37 AGA CAC GGA TGC AGA GTT TGA A GGG CGC CAT AGA TGC TTG TA
Fpn NM_016917 Solute carrier family 40, member 1 CTA CCA TTA GAA GGA TTG ACC AGC TA ACT GGA GAA CCA AAT GTC ATA ATC TG
Trf NM_133977 Transferrin TGG AGA CAG ATG CTC CCT CC TTT GTG CTC TGT GTA TGT GGT AAG G
Trfc NM_011638 Transferrin receptor TCA TGA GGG AAA TCA ATG ATC GTA GCC CCA GAA GAT ATG TCG GAA
Trfr2 NM_015799 Transferrin receptor 2 GCT GGT TCG GAC CTT CTC TTC AAC AAA AGA CTT CTT CGA GGT CTG A
Lcn2 NM_008491 Lipocalin 2 CAA GCA ATA CTT CAA AAT TAC CCT GTA GCA AAG CGG GTG AAA CGT T
Table S2. Analysis of iron metabolism gene expression in mice with severe anemic
hypoxia. Fold changes in iron metabolism gene expression in anemic controls, anemic
hepatocyte-specific Hif-1α and/or Hif-2α knock out mice compared to livers from
untreated normocythemic wild type controls (n=7). First column: livers of anemic
controls (controls; n=3); second column: livers, which lack hepatocyte-derived Hif-1α
(Hif1; n=3); third column: livers which lack hepatocyte-derived Hif-2α (Hif2; n=6);
fourth column: livers, which lack both Hif-1α and Hif-2α in hepatocytes (Hif1Hif2;
n=4). Abb.: VEGF, vascular endothelial growth factor; Cp, ceruloplasmin; Heph,
hephaestin; Aco1, aconitase; Ireb2, iron regulatory protein 2; Alas2, delta-
aminolevulinate synthase 2; Hamp, hepcidin; Hfe, hemochromatosis protein; Hfe2,
hemojuvelin; Dmt1, divalent metal transporter 1; Mfrn, mitoferrin; Fpn, ferroportin;
Trf, transferrin; Trfc, transferrin receptor 1; Trfr2, transferrin receptor 2; Lcn2,
lipocalin 2 / NGAL. Asterisks indicate statistically significant changes in gene
expression (unpaired Student’s t-test, P<0.05).
Figure S1. Characterization of P3Pro mutants. (A) Shown are serum iron (Tot. Fe)
and total iron binding capacity (TIBC) in P3Pro mutants and wild type controls (n=6 for
each genotype). (B) Shown are serum BUN (top panel) and creatinine (bottom panel)
levels in P3Pro and littermates controls (n=6 for each genotype). Measurements of BUN
and creatinine, as well as iron studies were performed by Anilytics, Gaithersburg, MD.
(C) To directly define whether bone marrow responsiveness is altered in mutant mice, we
examined the erythropoietic response after treatment with human recombinant EPO in
P3Pro mutants, wild type littermates and pre-conditioned normocythemic P3Pro mutants
(mice were exposed to prolonged hypoxia (10 % O2), which raised Hcts to wild type
levels). Human recombinant EPO (Procrit; Amgen, Thousand Oaks, CA) was
administered intraperitoneally, 3 times a week at a dose of 2000 units/kg body weight for
a total of four doses. After completion of treatment, we found no significant difference in
Hcts between the three groups, indicating normal bone marrow responsiveness in P3Pro
mutants. Shown are Hct values at baseline (day 0) and at day 10 of EPO treatment in
P3Pro mutants (n=4), wild type littermates (n=5) and preconditioned normocythemic
P3Pro mutants (n=4). (D) Genomic PCR analysis of DNA isolated from kidneys of mice
homozygous for the conditional Hif-2α allele at postnatal days (P) 2, 10, 20 and 90.
Primers used, amplify the conditional (2-lox) and the recombined (1-lox) allele: lane 1,
Cre-negative mouse; lanes 2 and 3, P3Pro mutants at P2; lanes 4 and 5, P3Pro mutants at
P10; lanes 6 and 7, P3Pro mutants at P20; and lane 8, P3Pro mutant at P90. ns, not
statistically significant.
Figure S2. Responses to PHD inhibition. (A) Top panel, shown are Hct values in
groups of five female BDF-1 mice dosed orally once per day with either vehicle (1%
methylcellulose) or GSK1002083A at 60, 30, 10, or 3 mg/kg for 8 consecutive days, n=5.
Bottom panel, serum Epo levels after oral administration of GSK1002083A for 2 days at
60 mg/kg in P3Pro mutants (n=3) and wild type littermate controls (n=4). (B) Western
blot analysis of Hif-1α and Hif-2α in kidney nuclear extracts from wild type mice and
P3Pro mutants at baseline conditions and after oral administration of GSK1002083A for
2 days at 60 mg/kg. Ponceau S staining is shown to demonstrate equal protein loading.
(C) Ldha (left panel) and Pgk (right panel) mRNA expression in mutant kidneys (n=3,
dark bars) compared to littermate controls (n=3, light grey bars) at baseline and after oral
administration of GSK1002083A for 2 days at 60 mg/kg. PHI, indicates treatment with
PHD inhibitor GSK1002083A. Bars represent mean values ± SEM; *, P<0.05; **,
P<0.01; ***, P<0.001.
Figure S3. (A) Quantitative analysis of iron levels (Tot. Fe) and total iron binding
capacity (TIBC) in serum from Albumin-Cre/Vhlh (VHL, n=5) and Albumin-Cre/Hif2α
mutant mice (Hif2, n=3) and wild type controls (Wt, n=4). Bars represent mean values ±
SEM.
Figure S4. Analysis of iron metabolism gene expression in mice with severe anemic
hypoxia. Shown are mRNA levels of iron metabolism genes (corresponds to table S2) in
livers from untreated control mice (first column, n=7), anemic controls (second column,
n=3), anemic Albumin-Cre/Hif1α knock out mice (third column, n=3), -Hif2a knock out
mice (fourth column, n=6) and anemic Albumin-Cre/Hif1α/Hif2α double knock out mice
(fifth column, n=4). Mean expression levels for untreated, normocythemic controls are
set to 1. Bars represent mean values ± SEM; * used for all levels of significance with
P<0.05.
P20 P90
P3ProCre-
Kidney
2-lox
1-lox
P10P2
Figure S1
0100200300400500
A B(u
g/dL
)
P3ProWt
Tot. Fe TIBC
nsns
P3Pro Wt
P3Pro Wt
ns
ns
BUN
Creatinine
10203040
0
0.00.10.20.30.40.5
(mg/
dL)
(mg/
dL)
C
Day 0 Day 10
Hct
(%)
01520
40
60
80
D
P3ProWtNormocythemic P3Pro
P3Pro P3ProWt Wt
Baseline PHI
0
2
4
6
Rel
. mR
NA
Ldha
/18S
Rel
. mR
NA
Pgk/
18S
BaselineWt WtP3Pro P3Pro
PHI
Hif-1α
Ponceau
A
C
*
****
***
*
ns**
0
2
1
3
P3Pro P3ProWt Wt
Baseline PHI
*
Hif-2α
P3Pro
Seru
m E
po x
100
0 (p
g/m
l)
1
10
100
VEH 10 360 3040
60
70
80
50
Hct
(%)
Wt
BFigure S2
GSK1002083A (mg/kg)
0
100
200
300
400
500
Tot. Fe TIBC
(ug/
dL)
Figure S3
Figure S4
Controls
Anemic Hif1 KO
Anemic Hif1Hif2 KO
Anemic controls
Anemic Hif2 KO
Epo Vegf Cp Heph
Aco1 Ireb2 Alas2 Hamp
Hfe Hfe2 Dmt1 Mfrn
Fpn Trf Trfc Trfr2
Lcn2