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Supplementary Info

Myeloid zinc finger 1 (MZF-1) suppresses prostate tumor growth

through enforcing ferroportin-conducted iron egress

Yue Chen1, 2, Zhihong Zhang1, Kuo Yang 1, Jin Du3, Yong Xu1,*, Sijin Liu2,*

1. Department of Urology, the Second Hospital of Tianjin Medical University,

Tianjin Institute of Urology, Tianjin 300211, China.

2. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

100085, China.

3. School of Life Sciences, Tsinghua University, Beijing 100084, China

Correspondence to Sijin Liu, Ph.D, or Yong Xu, MD

Email: sjliu@rcees.ac.cn

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Supplementary Figure Legends

S Fig. 1 Increased intracellular iron promotes PC3 cell growth. (A) Cell number

counting of PC3 cells after treatment with 200 μM Fe-NTA at different time points as

indicated (n=4). (B) The MTT assay indicated the cell viability upon 200 μM Fe-NTA

treatment at different time points as indicated (n=6).

S Fig. 2 MZF-1 regulates FPN expression in DU145 cells. (A) FPN and ferritin

concentrations in DU145 cells upon downregulation of endogenous MZF-1. (B)

Western blot of FPN and ferritin concentrations in DU145 cells with forced

expression of MZF-1.

S Fig. 3 MZF-1 regulates DU145 cell growth and cell viability. (A) Cell growth

reflected by cell number counting after knocking-down MZF-1 for 48h (n=4). (B)

Cell viability determined by the MTT assay upon knocking-down MZF-1 for 48h

(n=6). (C) Cell number counting upon forced expression of MZF-1 for 48h (n=4). (D)

Cell viability assessed by the MTT assay after 48-h transfection of MZF-1

overexpression plasmid (n=6).

S Fig. 4 DNA replication and intracellular ATP levels in DU145 cells. (A)&(B)

DNA replication was evaluated by the BrdU assay upon downregulation or forced

expression of MZF-1 for 48 h (n=6). (C)&(D) Relative ATP levels were assayed with

a microplate reader in DU145 cells in response to MZF-1 knockdown or

overexpression for 48 h (n=4).

S Fig. 5 Relative MZF-1 mRNA levels in PC3 cells upon miR-492 mimic molecule

transfection. qRT-PCR analysis of MZF-1 expression in PC3 cells after 48-h

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transfection of miR-492 mimic molecules (n=4).

S Fig. 6 miR-492 enhances PC3 cell proliferation. Cell proliferation assessed by the

BrdU incorporation assay after transfection of miR-492 mimic molecules for 48 h

(n=4).

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S Table 1. Predicted transcription factors that potentially regulate MZF-1

Gene Scroe Gene Score

HSF 100.0 Sn 93.1

AML-1a 100.0 HSF2 92.9

Lyf-1 100.0 GATA-2 92.2

c-Rel 99.2 Hb 92.0

NIT2 98.8 GC box 91.9

CdxA 98.6 STRE 91.5

ADR1 98.5 CREB 91.0

GATA-1 98.4 SRY 90.9

CDP CR 97.6 StuAp 90.8

Nkx-2 97.1 USF 90.8

MyoD 96.8 c-Myb 90.7

cap 96.2 GATA-3 90.5

AP4 94.6 Sp1 90.4

Dfd 93.4 STATx 90.4

Ttk 69 93.3 HNF-3b 90.2

NF-kap 93.1

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S Table 2. Predicted miRNAs that potentially regulate MZF-1

miRNA

miR-1228 miR-4292

miR-128 miR-452

miR-1291 miR-491-5p

miR-139-5p miR-492

miR-147b miR-581

miR-181a miR-591

miR-21 miR-600

miR-30b miR-615-3p

miR-3130-5p miR-661

miR-3165 miR-92a -2

miR-3187 miR-934

miR-3191 miR-98

miR-3194 let-7a

miR-330-5p let-7b

miR-363 let-7c

miR-3652 let-7d

miR-3681 let-7e

miR-3689a-3p let-7f

miR-380 let-7g

miR-4256 let-7i

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S Table 3. Oligonucleotide primers for knock-down and ChIP assays

Name Sequence (5’ -> 3’)

siRNA-MZF-1 pr1 Sense GAGGUCCUAUCAGAGAAGA

siRNA-MZF-1 pr1 Anti-Sense UCUUCUCUGAUAGGACCUC

siRNA-MZF-1 pr1 Sense CUACUGUAGGUGUCCAAUA

siRNA-MZF-1 pr1 Anti-Sense UAUUGGACACCUACAGUAG

siRNA-AP4 Sense GUGAUAGGAGGGCUCUGUAG

siRNA-AP4 Anti-Sense CUACAGAGCCCUCCUAUCAC

siRNA-c-Myb Sense AAGCACUUAAAGGGGAGAAUU

siRNA-c-Myb Anti-Sense UUCUCCCCUUUAAGUGCUUGG

MZF-1 Sense TGCAAGCCTCCGTTTGCTCCT

MZF-1 Anti-Sense CGGTCGCAGTGCAAAGCTCC

AP4 Sense ACCCAGACTCAGATCAGCAC

AP4 Anti-Sense CAGCTAAAGCAGACGGCTCG

c-Myb Sense CCCATCCTTCTCTCTCCCACA

c-Myb Anti-Sense TCCACCCAGACTTCGTGAGG

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S Table 4. Oligonucleotide primers for qRT-PCR analyses

Name Sequence (5’ -> 3’)

MZF-1 Sense AGTGTAAGCCCTCACCTCC

MZF-1 Anti-Sense GGGTCCTGTTCACTCCTCAG

AP4 Sense GCTTCTCAGAGTTGCTGTGC

AP4 Anti-Sense CAGCATCGGTTCTTCCTCTG

c-Myb Sense GCACCAGCATCAGAAGATGA

c-Myb Anti-Sense CTTTCCACAGGATGCAGGTT

GAPDH Sense GAAGGTGAAGGTCGGAGT

GAPDH Anti-Sense GAAGATGGTGATGGGATTTC

miR-98 Sense CGGCGGTGAGGTAGTAAGTTGTA

miR-147b Sense CGGCGGGTGTGCGGAAATGCTTC

miR-380 Sense CGGCGGTGGTTGACCATAGAACA

miR-452 Sense CGGCGGAACTGTTTGCAGAGGAA

miR-491 Sense CGGCGGAGTGGGGAACCCTTCCA

miR-492 Sense CGGCGGAGGACCTGCGGGACAAGA

microRNA Anti-Sense GTGCAGGGTCCGAGGT

U6 Sense GTGCTCGCTTCGGCAGCACA

U6 Anti-Sense GCAAATTCGTGAAGCGTTCC

S. Fig 1

Re

lati

ve

ab

so

rba

nc

e

48h

(A)

(B)

Re

lati

ve

ab

so

rba

nc

e

24h

0.5

1.0

1.5

0Cont Fe-NTA

P<0.001

Cont Fe-NTA

0.5

1.0

1.5

0

P=0.001

24h

0.25

0.75

1.0

0Cont Fe-NTA

P<0.001

Ce

ll n

um

be

r (×

10

5)

0.5

48h

Cont Fe-NTA

0.5

1.0

1.5

0

P<0.001

Ce

ll n

um

be

r (×

10

5)

2.0

S. Fig 2

MZF-1

FPN

Ferritin

GAPDH

(A) (B)

FPN

MZF-1

Ferritin

GAPDH

MZF-1-siRNAScrambled MZF-1-plasmidVector

MZF-1-siRNAScrambled

Re

lati

ve

ab

so

rba

nc

e

0.4

0.8

0

1.2 P<0.001

MZF-1-siRNAScrambled

1

0

1.5C

ell

nu

mb

er

(×10

5)

0.5

2P=0.024

Vector MZF-1-plasmid

P<0.001

Re

lati

ve

ab

so

rba

nc

e

0.4

0.8

0

1.2

Vector MZF-1-plasmid

1

0

1.2

Ce

ll n

um

be

r (×

10

5)

0.5

P=0.005

S Fig. 3

(A) (B)

(C) (D)

Scrambled

0.5

1

0

1.5

Brd

U i

nc

orp

ort

ati

on P<0.001

MZF-1-siRNA Vector MZF-1-plasmid

0.5

1

0

1.5

Brd

U i

nc

orp

ort

ati

on

P<0.001

0.5

1

0

1.5

AT

Pp

rod

ucti

on

MZF-1-siRNAScrambled

P=0.04

P=0.029

0.5

1

0

1.5

AT

P p

rod

ucti

on

Vector MZF-1-plasmid

S Fig. 4

(A) (B)

(C) (D)

S Fig. 5

MMNC miR-4920

2.5

2

1

0.5

1.5

MZ

F-1

mR

NA P=0.956

S Fig. 6

MMNC miR-492

0.5

1

0

1.5

Brd

U in

co

rpo

rtati

on

P=0.037