CD24

C

D

E

LacZ Snail1 Zeb2 Twist1

CD

44

Snail Twist1 Zeb2 LacZ

0.2 95.9 90.9 84.7

Twist1 Snail Zeb2 LacZ

** **

**

Snail Twist1 Zeb2 LacZ

Supplementary Figure S1. EMT promoting transcription factors induce stemness characteristics.

A, Overexpression of Foxq1 significantly induced EMT in HMLE cells. Left Panel: Morphological change.

Right panel: expression of EMT markers. B, Overexpression of Snail, Twist1 and Zeb2 significantly

induced EMT in HMLE cells. C, Overexpression of Snail, Twist1 and Zeb2 significantly increased the

CD44high/CD24low cell population in HMLE cells. D, Summary of the mammosphere formation assay for

Snail1, Twist1 and Zeb2 overexpressing HMLE cells (**P<0.01). E, Representative figures for the

mammosphere formation assay of Snail1, Twist1 and Zeb2 overexpressed HMLE cells. HMLE cells

transfected with LacZ was used as a control.

B

A

LacZ Foxq1

0.01

0.1

1

10

100

E-cadherin N-cadherin Vimentin Fibronection

LacZFoxq1

Rela

tive e

xpre

ssio

n

Num

ber

of

mam

mosphere

s

(per1

,000 c

ells

)

0

10

20

30

40

50

0 100 nM 2 nM 4 nM 50 nM Paclitaxel Doxorubicin

LacZ

F

oxq1

Cell types Numbers of implanted cells

20,000 9,000 3,000

4T1/NT 5/5 (100%) 5/5 (100%) 4/5 (80%)

4T1/sh3 5/5 (100%) 4/5 (80%) 1/5 (20%)

A B

C

Nu

mb

er

of

ma

mm

osp

he

res

(pe

r1,0

00

ce

lls)

4T1/NT 4T1/sh3 05

101520253035 **

Supplementary Figure S2. The role of Foxq1 in drug resistance, mammosphere formation and

tumor initiation. A, Representative images of the clonogenic assays of HMLE cells with or without

Foxq1 overexpression treated with different doses of doxorubicin (dox) or paclitaxel (pac). B, Knockdown

of Foxq1 significantly decrease mammosphere formation ability of 4T1 cells. Top panel: summary of

mammosphere formation results. Bottom panels: representative image of mammosphere formation. C,

Summary of tumor initiation capability of Foxq1 in 4T1 cells. Five mice were used for each group.

Percentiles represent tumor formation in specific groups. D, The effect of Foxq1 in apoptosis. Tumors

were collected from the 4T1 implanted BALB/C mice, and the prepared tissue slides were analyzed with

IHC using anti-BAX, p27 and Cleaved Caspase 8 antibodies, as well as H&E staining. The origins of all

four tumor samples are indicated at the top of the panels. NT: tumor from 4T1/NT cells bearing mouse.

SH3: tumor from 4T1/SH3 Foxq1 knockdown cell bearing mouse. NT+Pac: tumor from 4T1/NT cells

bearing mice treated with Paclitaxel. SH3+Pac: tumor from 4T1/Sh3 cells bearing mice treated with

Paclitaxel. Scale bar, 20 µm.

D NT SH3 NT+Pac SH3+Pac

p27

Cle

aved

Caspase

8

HE

B

AX

E

C

D

B A

***

**

Rela

tive e

xpre

ssio

n

***

Rela

tive e

xpre

ssio

n

**

**

0

4

6

8

2

10

0

4

6

8

2

10

Vector (μg)

Twist1(μg)

0.6 0 0.3

0 0.6 0.3

Rela

tive L

uc. activity

1.0

1.5

2.0

0.5

2.5

0

T-Pα2

**

**

T-Pβ1

***

***

0.6 0 0.3

0 0.6 0.3

Rela

tive L

uc. activity

2

0

4

3

1

Twist1 LacZ Zeb2 LacZ

F

Supplementary Figure S3. Twist1 and Zeb2 regulate PDGFRs. A and B, The relative expression level

of the Foxq1, Zeb2/Twist1 and PDGFRα and β genes in Twist1 (A) or Zeb2 (B) overexpressing HMLE cells was

measured by real-time RT-PCR assay (*P<0.05, **P<0.01 and ***P<0.001). C, Potential binding sites of Twist1

and Zeb2 in the promoter regions of PDGFRα and β genes were identified by an in silico analysis. Top and

middle panels show Twist1 binding sites in PDGFRα and β promoter region. Low panel shows Zeb2 binding site

in PDGFRβ promoter region. The conserved binding sites are highlighted with red. D, ChIP-qPCR analysis

shows that enrichment of one Twist1 binding site DNA from the PDGFRα and β promoter region, respectively

(**P<0.01). Dotted line represents 2-fold enrichment. However, ChIP-qPCR assay did not show enrichment of

DNA for Zeb2 binding in PDGFRβ promoter. E, Luciferase assay shows Twist1 activated the PDGFRα (left

panel) and β (right panel) gene promoter in a dose-dependent manner (**P<0.01, ***P<0.001). F, The binding of

Twist1 to the PDGFRα and β promoter region was confirmed by luciferase assay. The Twist1 conservative

binding sequence (WT) and mutant sequence (MT) for PDGFRα and β promoters was shown on the top of the

panels. The mutation of the binding sequence diminished the activation of both gene promoters by Twist1

(**P<0.01, ***P<0.001).

00.5

11.5

22.5

3

WT MT

Rela

tive L

uc. activity

CGCCACCTGCTG WT MT

T-Pβ1

CGCATCCCACTG

-6,000

PDGFR +1 -500 -1,000 -1,500 -2,000

T-Pα

1

T-Pα

2

T-Pα

3

T-Pα

4

T-Pα

5

PDGFRβ +1 -2,000

T-Pβ

1

T-Pβ

2

T-Pβ

5

T-Pβ

6

T-Pβ

7

T-Pβ

8

T-Pβ

9

-4,000

T-Pβ

10

T-Pβ

3

T-Pβ

4

PDGFRβ +1

Z-Pβ

1

Z-Pβ

5

Z-Pβ

6

Z-Pβ

2

Z-Pβ

3

Z-Pβ

4

+2,000 -2,000 -4,000

00.5

11.5

22.5

33.5

4

Twist1

LacZ

TCACACATGGAA WT MT TCAATCACAGAA

T-Pα2

WT MT

Rela

tive L

uc. activity

** ***

Twist1

LacZ

-Foxq1-/Twist1-/PDGFRs-

-Foxq1+/Twist1+/PDGFRs+

*

Supplementary Figure S4. Correlation and survival analysis of Foxq1 and Twist1 with PDGFRα

and PDGFRβ in tumor samples. A, The expression correlation of Foxq1 or Twist1 with PDGFRα and

β in breast cancer. Level 3 gene expression (RNAseV2) data of breast tumors from The Cancer

Genome Atlas (TCGA) database was used for the analysis. In the table, the upper panels (highlighted

in green) show correlation coefficients and the lower panels (highlighted in orange) include the

corresponding correlation p-values. B, The Kaplan-meier plot shows that overexpression of Foxq1 and

Twist1 with PDGFRs predicts poor survival of breast cancer patients in TCGA dataset (*P<0.05). C,

The expression correlation of Foxq1 or Twist1 with PDGFRα and β in Uterine corpus endometrial

carcinoma (UCEC). Level 3 gene expression (RNAseV2) data in UCEC from The Cancer Genome

Atlas (TCGA) database was used for the analysis. The table setting is same as in panel A.

Pvalue/correlation FOXQ1 Twist1 PDGFR-A PDGFR-B

FOXQ1 0.29 0.21 0.16

Twist 4E-14 0.45 0.47

PDGFR-A 4.6E-11 <2.2E-16 0.67

PDGFR-B 3.2E-4 <2.2E-16 <2.2E-16

Breast cancer of TCGA database A

B

Pvalue/Correlation FOXQ1 TWIST1 PDGFRA PDGFRB

FOXQ1 0.164 0.16 0.143

TWIST1 0.00227 0.375 0.32

PDGFRA 0.0431 2.33E-11 0.709

PDGFRB 0.0107 3.36E-10 0

C UCEC of TCGA database

Supplementary Figure S5. Effect of PDGFRα and PDGFRβ on cell migration, invasion and stem

cell characteristics. A-B, Knockdown of PDGFRα (A) and PDGFRβ (B) expression in HMLE /Foxq1

cells. Five different shRNAs specifically targeting PDGFRα or β were tested. β-actin was used as a

protein loading control. C-D, The effect of PDGFRs silencing on cell migration and invasion of

HMLE/Foxq1 cells. All stable cell lines with significant inhibition of PDGFRα and β expression showed

significant decrease in cell migration (C) (*P<0.05) and invasion (D) ( **P<0.01). E Effect of PDGFRs on

Foxq1 induced CD44high/CD24low cell population. Two PDGFRα knockdown cells showed minor cell

population changes, while two PDGFRβ knockdown cell models showed marked decrease of

CD44high/CD24low cell population. F, Summary of the effect of PDGFRα and PDGFRβ knockdown on

mammosphere formation of HMLE/Foxq1 cells (*P<0.05, **P<0.01). G, Representative figures of

mammosphere formation of HMLE/Foxq1 cells with PDGFRα and PDGFRβ knockdown. H,

Immunofluoresence assay showed no expression change of E-cadherin and Vimentin in HMLE/Foxq1

cells with individual and double knockdown of PDGFRα and β.

HMLE/Foxq1

α1 α5 β3 NT β4 HMLE/LacZ

H

E-c

adherin

V

imentin

HMLE/ LacZ NT 1 β4 1β4

HMLE/Foxq1

F

*

**

HMLE/Foxq1

α1 α5 β3 NT β4

*

HMLE/LacZ

0

40

60

20

80

Nu

mb

er

of

ma

mm

osp

he

res

(pe

r1,0

00

ce

lls)

-PDGFRα

α1 α2 α3 α4 α5 NT

-β-actin

-PDGFRβ

-β-actin

β1 β2 β3 β4 β5 NT A B

C D

Rela

tive

Invaded C

ells

β3 β4 NT α1 α5

** ** ** **

G

Rela

tive

Mig

rate

d C

ells

** *

β3 β4 NT α1 α5

** **

CD24

CD

44

HMLE/Foxq1

α1 α5 β3 NT β4

HMLE/LacZ

E

68.0 83.0 84.5 88.2 65.7 0.2

Supplementary Figure S6. Imatinib treatment on PDGFRs phosphorylation, mammosphere

formation and EMT. A, The expression of c-abl, kit and PDGFRs in HMLE cells with Foxq1 or

control LacZ overexpression was detected by real time RT-PCR (*P<0.05, **P<0.01). B,

Overexpression of Foxq1 leads to phosphorylation of PDGFRα and β, but not the c-ABL and c-kit. C,

imatinib (Ima) treatment inhibit phosphorylation of PDGFRs. Western blot analysis was performed

using total and phosphor-PDGFRα and β antibodies. D, Representative figures for mammosphere

formation in HMLE/Foxq1 cells after treatment of different doses of imatinib. E, No expression

changes for epithelial and mesenchymal markers in HMLE/Foxq1 cells with different doses of imatinib

treatment were detected by western blotting assay. F, Immunofluoresence assay shows no

expression change of E-cadherin and Vimentin proteins in HMLE/Foxq1 cells with different doses of

imatinib treatment.

B A

Rela

tive e

xpre

ssio

n

0 2.5 5

-β-actin

-PDGFRβ

-PDGFRα

-p-PDGFRβ

-p-PDGFRα

Ima (µM)

*

**

PDGFRα PDGFRβ c-ABL c-Kit 0

2

3

4

1

5

6

7 Foxq1 LacZ

C

-p-ABL

-p-Kit

-β-actin

-p-PDGFRβ

-p-PDGFRα

LacZ Foxq1

0 2.5 5

HMLE/Foxq1, Ima (µM) HMLE/ LacZ

0 2.5 5

HMLE/Foxq1, Imatinib (µM)

10

HMLE/LacZ

E-c

adherin

V

ime

ntin

D

E F

HMLE/Foxq1

Dox (nM)

C

Rela

tive s

urv

ived c

ells

Rela

tive s

urv

ived c

ells

***

B

** ***

** **

**

**

α1 β4 α1β4

NT

Pac (nM) 0 2 4

Rela

tive s

urv

ived c

ells

α1 β4 α1β4

NT

*

***

**

* **

***

Dox (nM) 0 40 80

NMuMG NMuMG D

Dox (nM) Pac (nM)

Rela

tive s

urv

ived c

ells

Rela

tive s

urv

ived c

ells

HMLER/Foxq1 HMLER/Foxq1

Ima 0 µM

Ima 5 µM

***

HMLE/Foxq1

Ima 0 µM

Ima 5 µM

Ima 0 µM

Ima 5 µM Ima 0 µM

Ima 5 µM

Pac (nM)

0

0.05

0.10

0.15

0.20 1.00

1.05

Rela

tive s

urv

ived c

ells

0

0.05 0.10 0.15 0.20

1.00 1.05

0.25 0.30 0.35 0.40

Supplementary Figure S7. The Effect of PDGFRs on chemoresistance. A and B, Chemoresistance

of HMLE/Foxq1 (A) and HMLER/Foxq1 (B) cells with individual or double PDGFRα and β knockdown

was analyzed by an MTT assay after treatment with various doses of doxorubicin left panel) and

paclitaxel (right panel). Results are presented as relative cell survival compared to the non-treatment

control (*P<0.05, **P<0.01 and ***P<0.001). C, Increased sensitivity of HMLE/Foxq1 cells to doxorubicin

(left panel) or paclitaxel (right panel) in the presence of imatinib (***P<0.001). D, NMuMG cells shows

no increased sensitivity to doxorubicin (left panel) and paclitaxel (right panel) in the presence of imatinib

(P>0.05).

HMLE/Foxq1

0 1 2

0.2

0.4

0.6

0.8

1.2

0

1.0

A

0 0.05 0.10 0.15 0.20

1.00 1.05

0.25 0.30

1.10 HMLE/Foxq1 R

ela

tive s

urv

ived c

ells

Dox (nM) 0 50 100

α1 β4 α1β4

NT

***

** **

***

*** ***

Rela

tive s

urv

ived c

ells

**

α1 β4 α1β4

NT

** ** **

** * **

Pac (nM)

Supplementary Figure S8. Immunohistochemistry (IHC) assays using tumor samples and our

working model. A, Tumors were collected from the HMLER/Foxq1 implanted NCR nu/nu mice, and

the prepared tissue slides were analyzed with IHC using anti-BAX, p27 and Cleaved Caspase 8

antibodies, as well as H&E staining. The origins of all four tumor samples are indicated at the top of

the panels. Scale bar, 50 µm. B, Tumors were collected from 4T1 implanted BALB/c mice. The other

settings are the same as panel A. C. Schematic model of the molecular mechanism underlying

Foxq1/PDGFR-driven breast cancer oncogenesis and chemoresistance. Foxq1, an EMT promoting

transcription factor, simultaneously regulates PDGFRα and β genes through direct or indirect

mechanisms. Twist1 and Zeb2 are the mediators of Foxq1’s indirect regulatory mechanism. This study

reveals the central regulatory role of Foxq1 in the TGFβ and PDGF signaling transition. Moreover, the

results of this study highlight PDGFR as important mediator of Foxq1/Twist1 promoted oncogenesis

and chemoresistance, which suggests an implication of designing novel combinational therapy for

breast cancer treatment (the dashed line indicates a relationship need further validation of in vivo

study).

Proliferation

Cell motility

Stemness

Chemoresistance

Oncogenesis

FOXQ1

ZEB2

PDGFRα

PDGFRβ

PD

GF

Rα

P

DG

FRβ

TG

F

β1

TG

Fβ

Rs

Nucle

us

Cyto

pla

sm

TWIST1

EMT

Control Ima Dox Ima+Dox

HMLER/Foxq1

p2

7

Cle

ave

d

Casp

ase

8

HE

B

AX

Control Ima Pac Ima+Pac

4T1

p2

7

Cle

ave

d

Casp

ase

8

HE

B

AX

A B

C