Epigenetic silencing of microRNA-203 is required for EMT and cancer stem cell

properties

Joseph H. Taube1,2, Gabriel G. Malouf3,4, Emily Lu5, Nathalie Sphyris1,2, Vidya Vijay1,2,

Priyanka P. Ramachandran1,2, Katumasa R. Ueno1, Sanchaika Gaur1, Milena S.

Nicoloso6, Simona Rossi6, Jason I. Herschkowitz7, Jeffrey M. Rosen7, Jean-Pierre J.

Issa8, George A. Calin6, Jeffrey T. Chang5,9, Sendurai A. Mani1,2, *

Figure S1 (Taube et al.)

a

b

miR-92b

HMLETwist

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e Ex

pres

sion

miR-375

HMLETwist

0.0

0.5

1.0

1.5

Rel

ativ

e Ex

pres

sion

miR-1247

HMLETwist

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e Ex

pres

sion

miR-1281

HMLETwist

0.0

0.5

1.0

1.5

Rel

ativ

e Ex

pres

sion

miR-203

miR-203

HMLETwist

0.0

0.5

1.0

1.5

Rel

ativ

e Ex

pres

sion

Figure S1 – Expression of other microRNAs with changes in promoter DNA methylation

Expression of indicated microRNAs was determined in HMLE control and HMLE Twist cells by

Taqman quantitative PCR. Levels were normalized to sno-U6.

Rel

ativ

e Ex

pres

sion

Vehicle 5azaC0.0

0.5

1.0

1.5

2.0

Rel

ativ

e Ex

pres

sion

Vehicle 5azaC0

5

10

15

Rel

ativ

e Ex

pres

sion

Vehicle 5azaC0

2

4

6

8

10

Rel

ativ

e Ex

pres

sion

Rel

ativ

e Ex

pres

sion

Vehicle 5azaC0.0

0.5

1.0

1.5

2.0

Rel

ativ

e Ex

pres

sion

Vehicle 5azaC0.0

0.5

1.0

1.5

2.0

miR-210

miR-92b miR-375 miR-1247miR-1281 miR-210

Rel

ativ

e Ex

pres

sion

HMLETwist

0

10

20

30

ns ns ns ns

nsnsnsnsns

Vehicle 5-AzaC0

5

10

15

p < 0.0001

p = 0.0212 p = 0.0058

D

A

Figure S2 (Taube et al.)

miR-20

3let

-7a

miR-92

a

miR-10

7

miR-12

6

miR-15

5

miR-20

0b

miR-22

4

miR-45

0a

miR-87

40.00001

0.0001

0.001

0.01

0.1

1

10

100 SUM159 ControlSUM159 miR-203

Fold

Cha

nge

Figure S2 – Expression of other microRNAs is unchanged by miR-203 over-expression

Relative levels of indicated microRNAs were quantified by Taqman quantitative PCR from RNA

extracts of control and miR-203 expressing SUM159 cells. Levels were normalized to sno-U6

and graphed relative to let-7a in control cells.

Figure S3 (Taube et al.)

a b

Figure S3 – miR-203 expression does not significantly affect cell growth rates

Cells were counted after the specified number of days in culture for both HMLE Twist (a) and

SUM159 (b) cells with either a control vector or miR-203.

Cell Growth in HMLE Twist

1 2 3 4 50

100

200

300

400 VectormiR-203

Days

Cel

ls x

10^

4

Cell Growth in SUM159

1 2 3 4 50

50

100

150VectormiR-203

DaysC

ells

x 1

0^4

Figure S4 (Taube et al.)

Hs578T

Control

miR-20

30

10

20

30

40M

amm

osph

eres

per

100

0 ce

lls

Figure S4 – miR-203 expression reduces mammosphere formation in Hs578t cells

Hs578t cells with either a control vector or expressing miR-203 were plated for mammosphere formation and spheres which formed after 10 days were counted.

p = 0.0017

a

Figure S5 (Taube et al.)

Control miR-2030

5

10

15

20

25%

CD

24+

cells

Figure S5 – miR-203 expression increases CD24 cell surface expression

CD24 cell surface expression was quantified by FACS in control vector and miR-203 expressing

SUM159 cells. The experiment was performed in triplicate.

p = 0.0146

SUM159 Control SUM159 miR-203

Figure S6

Figure S6 – Luciferase activity is statistically equivalent between control and miR-203

expressing SUM159 cells

SUM159 cells with either a control vector or expressing miR-203 were transduced with a

retrovirus expressing both RFP and luciferase and sorted for RFP expression. A 2-fold serial

dilution of cells was plated in a 6-well plate and luciferase activity was measured after adding

luciferin.

(Taube et al.)