0

20

40

60

80

100

120

140

160 PBS

DOX

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

OC

R (

pm

ol/m

in)/

ug

pro

tein

Control

DOX

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Lu

cif

era

se

(f

old

ch

an

ge

ove

r E

V)

y = 410.66x - 0.4014 R² = 0.997

0

20

40

60

80

100

120

0.0 0.1 0.2 0.3

Do

xo

rub

icin

(µ

M)

Absorbance (480 nm)

A B

baseline OM CCCP R/A

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control DOX

Ce

llu

lar

no

n-h

em

e iro

n

(μM

/μg

pro

tein

)

*

*

TfR1 Fpn1

*

0

0.5

1

1.5

2

2.5

Lu

cif

era

se

(fo

ld c

han

ge o

ver

EV

)

0

0.2

0.4

0.6

0.8

1

Lu

cif

era

se

(fo

ld c

han

ge

ov

er

EV

)

TfR1-IRE

Control TfR1-IRE

DOX TfR1-IRE∆

DOX

TfR1-IRE

Control TfR1-IRE

DFO TfR1-IRE∆

Control

TfR1-IRE∆ DFO

* * * * * *

* * *

*

* N.S.

C

E F G

H

N.S. m

RN

A l

ev

els

(% c

han

ge o

ver

co

ntr

ol)

D

0

2000

4000

6000

8000

10000

12000

Control DOX

Mean

TM

RE

flu

ore

scen

ce

*

Supplemental Figure 1. Doxorubicin regulates cellular iron homeostasis

(A) Absorbance (480 nm) was measured in NRCM at increasing concentrations of DOX. (B) Analysis of mitochondrial

oxygen consumption rate (OCR) in NRCM with DOX or control treatment quantified using Seahorse Bioanalyzer.

Respiration rates are measured at baseline, in the presence of oligomycin (OM, inhibitor of ATP synthase), carbonyl cyanide

m-chlorophenol (CCCP, dissipation of proton gradient), and rotenone/antimycin A (R/A, respiratory chain inhibitors) and

normalized to protein concentration of each sample (n=12). (C) Mitochondrial membrane potential assessed by TMRE

staining and flow cytometry in NRCM treated with 10μM DOX for 16 hours (n=3). (D) Total cellular non-heme iron levels in

NRCM treated with 10μM DOX for 16 hours determined by Ferene S assay (n=3). (E) mRNA expression of cellular iron

regulatory proteins, transferrin receptor 1 (TfR1) involved in iron import, and ferroportin 1 (Fpn1) involved in iron export, in

NRCM treated with 10μM DOX for 16 hours (n=6). (F-H) Luciferase assay of regulation of either full-length 3’UTR of TfR1

(TfR1-IRE) or 3’UTR of TfR1 with deletion of all five IREs (TfR1-IRE∆) by 200 μM DFO (F,G) and 10μM DOX (H) in HEK293

cells treated for 16 hours and normalized to renilla luciferase expression and empty vector (EV) expression (n=12). Data are

presented as mean±SEM. *P < 0.05.

0

0.2

0.4

0.6

0.8

1

1.2

ABCB6 ABCB7 ABCB10

mR

NA

leve

ls (f

old

chan

ge)

- DOX + DOXA B

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ABCB6 ABCB7 ABCB10

PBS DOX

mR

NA

leve

ls (F

old

chan

ge)

in vivo in vitro

0

20

40

60

80

100

120

control 0.1 µM 1 µM 10 µM 100 µM

0102030405060708090

100

Control 1µM 10 µM 100 µM0

0.2

0.4

0.6

0.8

1

1.2

Control 0.1µM 10 µM 100 µM

DAP

I neg

ativ

e

(Per

cent

sur

viva

l)

*

E

*

Anne

xin

V ne

gativ

e

(Per

cent

sur

viva

l)

D

Luci

fera

se a

ctiv

ity

(Rel

ativ

e to

con

trol

)

* *

F

0

20

40

60

80

100

120

0 μM 1 μM 10 μM 100 μM

Tryp

an b

lue

nega

tive

(P

erce

nt s

urvi

val)

*

C

Supplemental Figure 2. Doxorubicin does not regulate other mitochondrial ABC proteins and DOX regulation of ABCB8 is not through its effects on cell death (A) mRNA levels of three other mitochondrial ABC transporter genes were measured in mouse heart that had been treated with 6 mg/kg per day injection every third day for a total of four injections (Protocol 2) or saline (PBS) (n=4). Measurements were performed via qRT-PCR, normalized to 18S rRNA expression, and reported as a percentage of measurements obtained in the absence of DOX treatment. (B) mRNA levels of mitochondrial ABC proteins in NRCM treated with 10 µM DOX or control vehicle (n=6). (C-E) Cell death in HEK293 cells in response to various doses of DOX, as assessed by DAPI staining (n=6) (C), Annexin V staining (n=6) (D), and Trypan blue staining (n=3) (E). (F) Luciferase assay of the effects of DOX on the expression of luciferase construct driven by 6 kb of ABCB8 promoter (n=6). DOX at doses that did not affect cell death (i.e., 10 µM) still caused a reduction in the levels of ABCB8. Data are presented as mean±SEM. *P < 0.05.

A

02000400060008000

100001200014000

Control siRNA ABCB8 siRNA

Cyt

osol

ic ir

on

(CPM

/μg

prot

ein)

- DOX + DOX

B

0

500

1000

1500

Control siRNA ABCB8 siRNA

Nuc

lear

iron

(C

PM/μ

g pr

otei

n)

- DOX + DOX

D

GAPDH

Lamin A

Nucleus Cytosol

Lamin C

Supplemental Figure 3. Nuclear and cytosolic iron levels with ABCB8 siRNA with and without DOX in NRCM. (A) Levels of other mitochondrial ABC protein in NRCM with and without ABCB8 downregulation. (B) Western blot analysis of nuclear and cytosolic fraction purity in NRCM (n=3). (C, d) Nuclear (C) and cytosolic (D) iron with control and ABCB8 siRNA in the presence and absence of DOX (n=3). Data are presented as mean±SEM. *P < 0.05.

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

ABCB6 ABCB7 ABCB8 ABCB10

Fold

Cha

nge

ControlABCB8

*

siRNA siRNA

C

0

5000

10000

15000

20000

Ad-GFP Ad-ABCB8

Cyt

osol

ic ir

on

(CPM

/μg

prot

ein)

- DOX + DOX

0

500

1000

1500

Ad-GFP Ad-ABCB8

Nuc

lear

iron

(C

PM/μ

g pr

otei

n)

- DOX + DOX

A B

Supplemental Figure 4. Nuclear and cytosolic iron levels with ABCB8 overexpression with and without DOX in NRCM (A) Nuclear iron and (B) cytosolic iron with control and ABCB8 overexpression in the presence and absence of DOX (n=3). Data are presented as mean±SEM. *P < 0.05.

00.20.40.60.8

11.2

Liver Lung Kidney

ABC

B8

(den

sito

met

ry)

NTG ABCB8 TG

0

1

2

3

4

5

NTG ABCB8 TG

Cyt

osol

ic ir

on

(μM

/μg

prot

ein)

- DOX + DOX

01234567

NTG ABCB8 TG

Nuc

lear

iron

(μ

M/μ

g pr

otei

n)

- DOX + DOX

0

100

200

300

400

NTG ABCB8 TGU

IBC

(μg/

dL)

- DOX + DOX

1

10

100

1,000

10,000

100,000

1,000,000A

BC

B8

mR

NA

leve

ls

(% o

f NTG

) * ABCB8

Tubulin

TG NTG

A B TG NTG TG NTG TG NTG

Liver Lung Kidney

0

5

10

15

NTG ABCB8 TG

Lung

mito

iron

(μ

M/μ

g pr

otei

n)

- DOX + DOX

C

05

1015202530

NTG ABCB8 TG

Live

r mito

iron

(μ

M/μ

g pr

otei

n)

- DOX + DOX

D

0

5

10

15

20

NTG ABCB8 TG

Kid

ney

mito

iron

(μ

M/μ

g pr

otei

n)

- DOX + DOX

E

0

50

100

150

200

NTG ABCB8 TGSeru

m ir

on (μ

g/dL

)

- DOX + DOX

0100200300400500

NTG ABCB8 TG

TIB

C (μ

g/dL

)

- DOX + DOX

F

G H I J

*

* *

Supplemental Figure 5. ABCB8 expression and iron parameters in ABCB8 TG mice with and without DOX treatment (A) mRNA levels of ABCB8 in the hearts of NTG and ABCB8 TG mice (n=3). (B) ABCB8 protein levels in the lungs, livers, and kidneys of NTG and ABCB8 TG mice were evaluated via Western blot. Representative image is on the left, densitometry analysis is on the right (n=3). (C-D) Nuclear (C) and cytosolic (D) iron levels in NTG and ABCB8 TG mice with and without DOX treatment (n=4). (E-G) Mitochondrial iron levels in the lung (E), liver (F) and kidney (G) of TG and NTG mice with and without DOX (n=3). (H-J) Hematologic iron parameters, serum iron (H), unsaturated iron binding capacity, UIBC (I), and total iron binding capacity, TIBC (J) in TG and NTG mice with and without DOX (n=4-5). Data are presented as mean±SEM. *P< 0.05.

A

0

10

20

30

40

% F

S

0

1

2

3

CO

/BW

(ml/m

in·g

) B C

0

20

40

60

80

100

120

ESP EDP

Pres

sure

(mm

Hg)

NTGTG

0

100

200

300

400

NTG TG

HR

(bpm

)

E

0

1

2

3

4

5

6

7

HW

/BW

(mg/

g)

100 μm

Hematoxylin & Eosin Masson Trichrome

I

0

1

2

3

4

5

LVD

d (m

m)

D

TG NTG NTG TG

TG TG

TG TG

F

NTG ABCB8 TG

G H

-6000

-4000

-2000

0

2000

4000

6000

8000

dP/d

t (m

mH

g/s)

NTGTG

dp/dtmax

dp/dtmin

NTG NTG NTG NTG

Supplemental Figure 6. Cardiac features of ABCB8 TG and NTG mice at baseline in the absence of DOX treatment M-mode echocardiographic images (A) were evaluated for measurements of fractional shortening (FS) (B), the ratio of cardiac output to body weight (CO/BW) (C), and left-ventricular diastolic diameter (LVDd) (D) in the hearts of NTG mice and ABCB8 TG (TG) mice. (E) Heart weights (n=5), and (F-G) invasive hemodynamic measurements of end systolic pressure (ESP) and end diastolic pressure (EDP) (F), dP/dtmax (a marker of systolic function) and dP/dtmin (a marker of diastolic function) (G), and heart rate (H) were performed in NTG mice and ABCB8 TG mice (n=4). (I) H & E-stained and Masson trichrome-stained sections of heart tissue from NTG mice and ABCB8 TG mice were evaluated for histological abnormalities. Data are presented as mean±SEM. *P< 0.05.

Control

DOX

Dox

0 3 6

PBS Analysis

37 day

Control

DOX

Protocol 1

Protocol 2

0 2 4 11 day

9

One week

One month

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11

% o

f sur

viva

l

Days after doxorubicin injection

NTG

mABC1 TG

B C

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35

% o

f sur

viva

l

Days after doxorubicin injection

NTG

mABC1TGTG

TG

A

0

100

200

300

400

500

NTG ABCB8TG

NTG ABCB8TG

PBS DOX

HR

0

10

20

30

40

NTG ABCB8TG

NTG ABCB8TG

PBS DOX

% F

S

* * *

% E

F

0

10

20

30

40

50

60

70

NTG ABCB8TG

NTG ABCB8TG

PBS DOX

D E F

Supplemental Figure 7. Survival rates and cardiac features of NTG and ABCB8 TG mice after DOX administration (A) Schematic representation of DOX treatment protocols. Protocol 1 consisted of a 10 mg/kg per day injection administered every other day for a total of three injections, and Protocol 2 used a 6 mg/kg per day injection every third day for a total of four injections. Cardiac function was assessed one week after the last injection for Protocol 1 and one month after the last injection for Protocol 2. (B,C) The survival rates of NTG and ABCB8 TG mice were monitored after treatment with 3 intraperitoneal injections of 10 mg/kg DOX (cumulative dose: 30 mg/kg) administered every other day (n=11) (B) or 4 intraperitoneal injections of 6 mg/kg DOX (cumulative dose: 24 mg/kg) administered every third day (n=7) (C). (D) Assessments of heart rate (HR) in NTG and ABCB8 TG mice after DOX treatment according to protocol 2 (n=6-8). (E,F) NTG and ABCB8 TG mice were treated with DOX via Protocol 1. One weeks after the final DOX injection (as opposed to one month later for Protocol 2), echocardiographic assessments of fractional shortening (FS) (E), and ejection fraction (EF) (F) were measured (n=3-5 per group). Data are presented as mean±SEM. *P< 0.05.

0

5000

10000

15000

Control DXZ DFO

Cyt

osol

ic ir

on

(CPM

/μg

prot

ein)

- DOX + DOX

0200400600800

100012001400

Control DXZ DFO

Nuc

lear

iron

(C

PM/μ

g pr

otei

n)

- DOX + DOX

*

#

A B

00.5

11.5

22.5

33.5

4

Control DOX DOX+DFO DOX+DXZ

Cyt

osol

ic ir

on

(μM

/μg

prot

ein)

C

0

1

2

3

4

5

6

Control DOX DOX+DFO DOX+DXZ

Nuc

lear

iron

(μ

M/μ

g pr

otei

n)

D

0

100

200

300

400

500

600

Hea

rt R

ate

DOX DOX +DXZ

DOX +DFO

Control

E

* * *

Supplemental Figure 8. Cytosolic and nuclear iron in mice treated with DFO and DXZ (A, B) Cytosolic (A) and nuclear (B) iron content in NRCM treated with 200μM DFO or 200μM DXZ for 16 hours with and without DOX in NRCM (n=3). (C,D) Cytosolic (D) and nuclear (D) iron levels in the hearts of WT mice treated with DOX, with or without DFO or DXZ (n=3-4). (E) Heart rate of control mice and DOX-treated mice that had been co-treated with or without DXZ or DFO. DOX treatment was according to protocol 2 (n=6).

Original Western Blots

ABCB8 protein levels in NRCMs With Dox treatment (20 mM DOX)

Full Unedited Gels for Figure 2A

Full Unedited Gels for Figure 2B

97

64

51

39

28

19

Control Control DOX DOX 97

64

51

39

28

19

ABCB8 Actin

Full Unedited Gels for Figure 2H

Mitochondrial Ferritin Tubulin

188

98

62

49

38

28

17

14

Full Unedited Gels for Figure 3A

97

64

51

39

28

19

GAPDH ABCB8

Control siRNA ABCB8 siRNA

Full Unedited Gels for Figure 5A

97

64

51

39

28

19

97

64

51

39

28

19

Full Unedited Gels for Figure 9A

188

98

62

49

38

28

17

14

6

Topoisomerase 2β and Tubulin ABCB8

Full Unedited Gels for Figure 9D

GAPDH

Top 2β

191

105

64

51

39

28

19

14

Full Unedited Gels for Figure 9E

188

98

62

49

38

28

17

14

6

Topoisomerase 2β and Tubulin

Full Unedited Gels for Figure 9F

188

98

62

49

38

28

17

14

Topoisomerase 2β Tubulin

Full Unedited Gels for Figure 9F (Cont)

Full Unedited Gels for Supplemental

Figure 3A

Lamin A/C GAPDH

188

98

62

49

38

28

17

14

Full Unedited Gels for Figure S5B

97

64

51

39

28

19

97

64

51

39

28

19