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