0

2

4

6

8

10

12

14

16

18

20

PPR

E-TK

-Luc

activ

ity

(fold

indu

ctio

nl)

PPARγ +RosiPGC-1β

SRC-1

PPRE-Tk-Luc

*

*

*

#

#

Supplementary materialFig 1

- - - - + + + +

- + - - - + - -

PGC-1α - - + - - - + -

- - - + - - - +

0

10

20

30

2mU

CP3

-Luc

activ

ity

(fold

indu

ctio

nl)

GR + dex

PGC-1β

SRC-1

2mUCP3-Luc

*

*

*#

#

- - - - + + + +

- + - - - + - -

PGC-1α - - + - - - + -

- - - + - - - +

*

*#

0

1

2

3

4

- + + +

PTK

-Luc

activ

ity(fo

ldin

duct

ion)

- - + -- - - +

pTk-Luc + e2F

DP1

PCAF

PCAF∆HAT

0

5

10

15

20

25

30

35

- + + +

2mU

CP3

-Luc

activ

ity(fo

ldin

duct

ion)

- - + -- - - +

2mUCP3-Luc

GR+dex

PCAFPCAF∆HAT

* **

*#

Supplementary materialFig 2

**

- +

AR

E-K

AP-

Luc

activ

ity

(fold

indu

ctio

n)

ARE-KAP-Luc+hARP + DHT

Tip-60

0

5

10

15

20

25

30

35

- +

2mU

CP3

-Luc

act

ivity

(fo

ld in

duct

ion)

2mUCP3-Luc+ GR + dex

Tip-60

Supplementary materialFig 3

0

1,0

2.0

(fold

indu

ctio

n) #

Supplementary materialFig 4

0

2

4

6

8

10

12

14

16

18

20

CR

E-PG

C1-

Luc

activ

ity

(fold

indu

ctio

nl)

- + - -

- - + -

SRαααα-PKA

SIRT3

CRE-PGC1-Luc 2mUCP3-Luc

*

*

0

4

8

12

16

20

24

28

32

40

2mU

CP3

-Luc

act

ivity

(fo

ldin

duct

ionl

)

- + - -

- - + -

GR + dex

SIRT3

36

# **

Figure S1.

SRC-1, PGC-1αααα and PGC-1ββββ do not co-activate the action of glucocorticoids on

mouse UCP3 promoter activity (2mUCP3) despite doing so on the PPARγγγγ-

dependent regulation of a PPAR-responsive element-driven luciferase reporter

gene in L6 myogenic cells. L6 myogenic cells were transiently transfected with 1.5 µg

of the apoAII-PPRE-TKpGL3 plasmid (PPRE-Tk-Luc) (a gift from Dr.L.Fajas,

University of Montpellier, France) and treated with 10 µM rosiglitazone. When

indicated 0.3 µg of pSV-PGC-1α (a gift from Dr. B. Spiegelman, Dana-Farber Cancer

Institute and the Department of Cell Biology, Harvard Medical School, MA, USA),

pcDNA3-PGC-1β (a gift from Dr. Vidal-Puig, University of Cambridge, UK) or

pcDNA3-SRC1 expression vector (a gift of Dr. S.Kato, Fuji Gotemba Research

Laboratories, Japan) were co-transfected. In parallel, the 2mUCP3-Luc vector, in which

the 2kb 5’ region of the mouse UCP3 gene drives the luciferase reporter gene, was

transfected to L6 cells and co-trasfected with the GR expression vector and exposed to

dexamethasone as described in the Materials and Methods section. Co-transfection with

the PGC-1α , PGC-1β and SRC-1 expression vectors was also performed. Assessment

of luciferase activity and calculations were performed as described in Material and

Method section. Results are means + SEM of at least 3 independent experiments.

Statistically significant differences due to PPARγ + rosiglitazone or to GR +

dexamethasone in each promoter are shown as *P<0.05, and those due to the co-

transfection of PGC-1α, PGC-1β or SRC1 expression vectors are shown as #P<0.05.

Data indicate that co-transfected SRC-1, PGC-1α and PGC-1β show co-activating

activity on a a PPAR-responsive element used as a positive control (1,2) but not on the

glucocorticoid-mediated induction of the UCP3 promoter activity.

Figure S2

PCAF does not co-activate the action of glucocorticoids on mouse UCP3 promoter

activity (2mUCP3) despite doing so on pTK-driven luciferase reporter gene in L6

myogenic cells.

L6 myogenic cells were transiently transfected with 3 µg of pTK-Luc ( a luciferase

expression vector driven by thymidine kinase promoter) and co-transfected with

pcDNA3-e2F1 (a gift of Dr. Martinez-Balbas, CSIC, Barcelona, Spain). When indicated

0.1µg of pCMV-DP1, and 0.3 µg of pcx-P/CAF or pcx P/CAF∆HAT, expression

vectors for the the wild-type and the acetylating-devoid form of P/CAF, respectively,

were co-transfected. In parallel, the 2mUCP3-Luc vector, in which the 2kb 5’ region of

the mouse UCP3 gene drives the luciferase reporter gene, was transfected to L6 cells,

co-transfected with the GR expression vector and exposed to dexamethasone as

described in the Material and Method section Assessment of luciferase activity and

calculations were performed as described in Material and Method section. Results are

means + SEM of at least 3 independent experiments Statistically significant differences

due to DP1 or to GR + dexamethasone in each promoter are shown as *P<0.05. and

those due to the co-transfection of P/CAF expression vectors are shown as #P<0.05.

Data indicate that co-tranfected P/CAF but not P/CAF∆HAT shows co-activation of the

DP1/e2F action on the TK promoter activity, used as a positive control (3). There was

no effect of P/CAF on the glucocorticoid-mediated induction of the UCP3 promoter

activity.

Figure S3

Tip-60 does not co-activate the action of glucocorticoids on mouse UCP3 promoter

activity (2mUCP3) despite doing so on the action of androgens on the KAP

promoter-driven luciferase reporter gene in L6 myogenic cells.

L6 myogenic cells were transiently transfected with 1.5 µg of ARE-KAP-Luc (a

luciferase expression vector driven by the androgen responsive element of the mouse

kidney androgen-regulated protein gene (4)), co-transfected with the expression vector

for androgen receptor (hARP), gifts from Dr.A.Meseguer (Institut de Recerca de la Vall

d’Hebron, Barcelona, Spain) and exposed to 10 µM dihydrotestosterone. When

indicated, 0.3 µg of the expression vector of Tip60 was co-transfected. In parallel, the

2mUCP3-Luc vector, in which the 2kb 5’ region of the mouse UCP3 gene drives the

luciferase reporter gene, was transfected to L6 cells and co-transfected with the GR

expression vector and exposed to dexamethasone as described in the Material and

Method section. Assessment of luciferase activity and calculations were performed as

described in Material and Method section. Results are means + SEM of at least 3

independent experiments. Statistically significant differences due to the co-transfection

of Tip60 expression vector is shown as #P<0.05. Data indicate that co-transfected

Tip60 shows co-activating activity on the androgen receptor-mediated induction of the

ARE-driven promoter activity used as a positive control (4,5). There was no effect of

Tip60 on the glucocorticoid-mediated induction of the UCP3 promoter activity.

Fig S4

SIRT3 does not affect the action of glucocorticoids on mouse UCP3 promoter

activity (2mUCP3) despite being capable of activating a protein kinase-A

responsive-element driven promoter in L6 myogenic cells.

L6 myogenic cells were transiently transfected with 3 µg of CRE-PGC1-Luc (a

luciferase expression vector containing the cAMP responsive element of the PGC-1α

(6) and co-transfected with 0.3 µg of the expression vector of the constitutively active

form of protein kinase-A (SRα-PKA) (7), a gift from Dr. M Muramatsu (DNAX

Research Institute of Molecular and Cellular Biology, CA, USA). When indicated, 0.3

µg of the expression vector of SIRT3 was co-transfected. In parallel, the 2mUCP3-Luc

vector, in which the 2kb 5’ region of the mouse UCP3 gene drives the luciferase

reporter gene, was transfected to L6 cells and co-transfected with the GR expression

vector and exposed to dexamethasone as described in the Material and Method section.

Assessment of luciferase activity and calculations were performed as described in

Material and Method section. Results are means + SEM of at least 3 independent

experiments. Statistically significant differences due to PKA or to GR +

dexamethasone in each promoter are shown as *P<0.05. and those due to the co-

transfection of SIRT3 expression vector are shown as #P<0.05. Data indicate that co-

transfected SIRT3 enhanced the PKA-dependent activity of the CRE-PGC1-Luc vector

used as a positive control (8) whereas there was no effect of SIRT3 on the

glucocorticoid-mediated induction of the UCP3 promoter activity.

References to Supplementary material 1. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV.(1998) Nature 395:137-43 2.Meirhaeghe A, Crowley V, Lenaghan C, Lelliott C, Green K, Stewart A, Hart K, Schinner S, Sethi JK, Yeo G, Brand MD, Cortright RN, O'Rahilly S, Montague C and Vidal-Puig AJ (2003) Biochem J 373:155-65. 3.Martinez-Balbas MA, Bauer UM, Nielsen SJ, Brehm A, Kouzarides T. (2000) EMBO J 19:662-71. 4.Soler M, Tornavaca O, Sole E, Menoyo A, Hardy D, Catterall JF, Vandewalle A, Meseguer A. (2002) Biochem J 366:757-66. 5.Brady ME, Ozanne DM, Gaughan L, Waite I, Cook S, Neal DE, Robson CN. (1999) J Biol Chem. 274:17599-604. 6.Hondares E, Mora O, Yubero P, Rodriguez de la Concepcion M, Iglesias R, Giralt M, Villarroya F. (2006) Endocrinology 147:2829-38. 7.Muramatsu M, Kaibuchi K, Arai K(1989)Mol Cell Biol 9:831-6 8.Shi T, Wang F, Stieren E, Tong Q (2005) J Biol Chem; 280:13560-7.