Supplementary Fig. 1. Analysis of beiging, mitochondrial and adipogenic markers in EpiWAT and BAT from Tgr5+/+ and Tgr5-/- mice exposed to cold. (A) Quantitative densitometry of the Western blots depicted in Figure 1h. (B and C) mRNA expression levels of beige remodeling and mitochondrial markers Pgc1a, Ucp1, Tbx1, Prdm16, Cidea, Cd137, Eva1, Dio2, Cpt1a, Pparg2 and Cebpb in the EpiWAT (B) and BAT (C) of TGR5 wild-type (Tgr5+/+) and germline TGR5 knock-out (Tgr5-/-) mice exposed to cold (8°C) for 7 days. n = 10 per group. (D and F) Representative (n = 10 per group) Western blot of PGC-1α, the mitochondrial marker VDAC1, and beiging markers TBX1 and UCP1 from the EpiWAT (D), and PGC-1α, PPARα, UCP1 and the mitochondrial marker VDAC1 from the BAT (F) of the mice described in B and C. GAPDH was used as loading control. (E and G) Quantitative densitometry of the Western blots showed in D (EpiWAT) and F (BAT). (H) Representative (n = 10 per group) Western blot of mitochondrial OXPHOS complexes (CII to CV) from the EpiWAT and BAT of the mice described in B and C. (I and J) Representative (n = 5 per group) hematoxylin and eosin stainings of EpiWAT (I) and BAT (J) sections from mice described in A and B. Scale bars = 50µm. Results represent mean ± SEM. * P ≤ 0.05 vs. Tgr5+/+ group by Student’s t-test.

Tgr5+/+ Tgr5 -/-

EpiWAT BAT

Tgr5+/+ Tgr5 -/-

CIV

CVCIII

CII

Tgr5+/+

Tgr5-/-

Tgr5+/+

Tgr5-/-

EpiWAT BAT

B

C

F G H

I J

PGC-1αPPARαUCP1

Tgr5+/+

Tgr5-/-

BAT

VDAC1GAPDH

PGC-1αTBX1UCP1VDAC1GAPDH

EpiWAT

PGC-1α/GAPDH

0.00.10.20.30.40.5

0.0

0.5

1.0

1.5

2.0

TBX1/GAPDH

PGC-1α/GAPDH

0.0

0.5

1.0

1.5PPAR

α/GAPDH

0.0

0.3

0.6

0.9

1.2

0.0

0.5

1.0

1.5

2.0

UCP1/GAPDH

0.00.51.01.52.02.5

VDAC1/GAPDH

0.0

0.5

1.0

1.5

Rel

ativ

e m

RN

A le

vels

Pgc1aUcp1

Eva1Prdm16

CideaDio2

Pparg2Cpt1a

** *

A

0

1

2

3

4

Complexes/GAPDH

p=0.06*

CIVCV CIII CII

0.0

0.5

1.0

1.5

Rel

ativ

e m

RN

A le

vels

*

Pgc1aUcp1

Tbx1Prdm16

CideaCd137

Pparg2Cebpb

*

D ETgr5

+/+

Tgr5-/-

0.00.10.20.30.40.5

VDAC1/GAPDH

37

37

37

50

100

37

37

37

50

100

25

37

50

Tgr5 +/+ Tgr5 -/-

Supplementary Fig. 2. Validation of WAT specific TGR5 knock-out (Tgr5Adipoq-/-) mice. (A to C) TGR5 mRNA expression levels in the indicated tissues of WAT specific TGR5 knock-out (Tgr5Adipoq-/-) mice and their controls (Tgr5Adipoq+/+) (n = 10 per group). Results represent mean ± SEM. ** P ≤ 0.01 and *** P ≤ 0.001 vs. Tgr5Adipoq+/+ group by Student’s t-test.

A

B

C

0.0

0.5

1.0

1.5

Kidney

Tgr5

mR

NA

leve

ls

0.0

0.5

1.0

1.5

Liver

Tgr5

mR

NA

leve

ls

0.0

0.5

1.0

1.5

2.0

HeartTg

r5 m

RN

A le

vels

0.0

0.5

1.0

1.5

Adrenal gland

Tgr5

mR

NA

leve

ls

0.0

0.5

1.0

1.5

Hypothalamus

Tgr5

mR

NA

leve

ls

0.0

0.5

1.0

1.5

BAT

Tgr5

mR

NA

leve

ls

Tgr5 Adipoq+/+ Tgr5 Adipoq-/-

0.0

0.5

1.0

1.5

EpiWAT

Tgr5

mR

NA

leve

ls

***

0.00.20.40.60.81.0

ScWAT

Tgr5

mR

NA

leve

ls ****

Supplementary Fig. 3. Analysis of beiging, mitochondrial and adipogenic markers in EpiWAT and BAT form Tgr5Adipoq+/+ and Tgr5Adipoq-/- mice exposed to cold. (A and B) mRNA expression levels of beige remodeling and mitochondrial markers Pgc1a,Ucp1, Tbx1, Prdm16, Cidea, Cd137, Eva1, Dio2, Cpt1a, Pparg2 and Cebpb in theEpiWAT (A) and BAT (B) of control mice (Tgr5Adipoq+/+) and WAT specific TGR5 knock-out (Tgr5Adipoq-/-) mice exposed to cold (8°C) for 7 days. n = 10 per group. (C and E)Representative (n = 10 per group) Western blot of PGC-1α, the mitochondrial markerVDAC1, and beiging markers TBX1 and UCP1 from the EpiWAT (C) and PGC-1α,PPARα, UCP1 and the mitochondrial marker VDAC1 from the BAT (E) of the micedescribed in A and B. GAPDH was used as loading control. (D and F) Quantitativedensitometry of the Western blots showed in C (EpiWAT) and E (BAT). (G and H)Representative (n = 5 per group) hematoxylin and eosin stainings of EpiWAT (G) andBAT (H) sections from mice described in A. Scale bars = 50µm. Results represent mean± SEM. * P ≤ 0.05 vs. Tgr5Adipoq+/+ group by Student’s t-test.

A

B

C

E F

G H

D

0.0

0.5

1.0

1.5R

elat

ive

mR

NA

leve

ls

Pgc1aUcp1

Eva1Prdm16

CideaDio2

Pparg2Cpt1a

0.0

0.5

1.0

1.5

Rel

ativ

e m

RN

A le

vels

Pgc1aUcp1

Tbx1Prdm16

CideaCd137

Pparg2Cebpb

0.00.10.20.30.40.5

PGC-1α/GAPDH

0.0

0.5

1.0

1.5

TBX1/GAPDH

0.0

0.1

0.2

0.3

0.4

UCP1/GAPDH

0.0

0.5

1.0

1.5

VDAC1/GAPDH

0.0

0.5

1.0

1.5

PGC-1α/GAPDH

0.0

0.5

1.0

1.5

PPAR

α/GAPDH

0

1

2

3

4

UCP1/GAPDH

Tgr5 Adipoq+/+

Tgr5 Adipoq-/-

PGC-1αTBX1UCP1VDAC1GAPDH

EpiWAT

Tgr5Adipoq+/+

Tgr5Adipoq-/-

PGC-1α

UCP1VDAC1GAPDH

BAT

PPARα

EpiWAT

Tgr5 Adipoq+/+ Tgr5 Adipoq-/-

BAT

Tgr5 Adipoq+/+ Tgr5 Adipoq-/-

0.00.51.01.52.02.5

VDAC1/GAPDH

*Tgr5Adipoq+/+

Tgr5Adipoq-/-

37

37

37

50

100

37

37

37

50

100

Supplementary Fig. 4. TGR5 activation promotes beiging in the scWAT at thermoneutrality and after high fat diet feeding. (A) Quantitative densitometry of the Western blots showed in Figure 3e. (B) Quantitative densitometry of the Western blots showed in Figure 4c (n = 10 per group). Results represent mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 vs. Tgr5+/+ + Vehicle control group by one-way ANOVA followed by Bonferroni post-hoc test (A); or Tgr5+/+ HF control group by Student’s t-test (B).

A

B

PGC

-1α

/GAP

DH

*

0.0

0.5

1.0

1.5

0.0

0.2

0.4

0.6

0.8

TBX1

/GAP

DH ***

0.00.20.40.60.81.0

UC

P1/G

APD

H ***

0.0

0.5

1.0

1.5

2.0

VDAC

1/G

APD

H

*

Tgr5 +/+ + INT-777 Tgr5 -/- + INT-777Tgr5 +/+ + Vehicle Tgr5 -/- + Vehicle

PGC

-1α

/GAP

DH

0.0

0.3

0.6

0.9

1.2 *

0.0

0.5

1.0

1.5

TBX1

/GAP

DH

**

0.0

0.5

1.0

1.5

2.0

UC

P1/G

APD

H ***

0.0

0.5

1.0

1.5

2.0

VDAC

1/G

APD

H ***

Tgr5 +/+ HF + INT-777Tgr5 +/+ HF

Supplementary Fig. 5. TGR5 activation promotes beige adipocyte differentiation in vitro. (A and B) mRNA expression levels of Tgr5, and beige remodeling markers Pgc1a, Ucp1, Cidea, Pparg2 and Cebpb in differentiated 3T3-L1 cells in presence of the TGR5 agonists INT-777 (A), lithocholic acid (LCA) (B) or vehicle (DMSO). 3T3-L1 cells were transfected with either shRNA for TGR5 (sh-TGR5), control shRNA (sh-Co) or mouse TGR5 (mTGR5), as indicated in the Figure. n = 6 (C) Representative (n = 6 per group) images of UCP1 immunofluorescence (in green) on the cells described in A and B. Nuclei were stained with DAPI (in blue). Scale bars = 50μm. (D) Quantification of fluorescent intensity showed in C. Normalization of fluorescence was performed with cell count obtained by nuclei (DAPI) staining. (E) Representative (n = 6 per group) images of TOMM20 immunofluorescence (in green) of adipocytes differentiated from the stromal vascular fraction (SVF) of TGR5 wild-type (Tgr5+/+) and germline TGR5 knock-out (Tgr5-/-

) mice. SVF cells were differentiated for 7 days in presence or absence of the TGR5 agonist INT-777. n = 6. Nuclei were stained with DAPI (in blue). Scale bars = 25μm (F) Quantification of fluorescent intensity showed in E. Results represent mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 vs. DMSO control group by Student’s t-test (A and B), or by one-way ANOVA followed by Bonferroni post-hoc test (D and F).

A

B C

DMSO INT-777 LCA

mTGR5sh-Co

sh-TGR5012

50

100Tg

r5 m

RN

A le

vels

*

0

1

2

3

4

Pgc1

a m

RN

A le

vels

mTGR5sh-Co

sh-TGR50

2

4

6

8

Ucp

1 m

RN

A le

vels *

*

mTGR5sh-Co

sh-TGR50.0

0.5

1.0

1.5

2.0

Cid

ea m

RN

A le

vels *

***

mTGR5sh-Co

sh-TGR5012345

Ppar

g2 m

RN

A le

vels

****

mTGR5sh-Co

sh-TGR5

P=0.08

***

mTGR5sh-Co

sh-TGR50

2

4

6

8

Ceb

pb m

RN

A le

vels

012

50

100

Tgr5

mR

NA

leve

ls

mTGR5sh-Co

sh-TGR50

1

2

3Pg

c1a

mR

NA

leve

ls

mTGR5sh-Co

sh-TGR5

*

*

0

1

2

3

4

Ucp

1 m

RN

A le

vels

*

mTGR5sh-Co

sh-TGR5

0

1

2

3

Cid

ea m

RN

A le

vels

*****

mTGR5sh-Co

sh-TGR50.0

0.5

1.0

1.5

Ppar

g2 m

RN

A le

vels

mTGR5sh-Co

sh-TGR5mTGR5

sh-Co

sh-TGR50

1

2

3

Ceb

pb m

RN

A le

vels

UCP1 DAPI MERGE

DMSO

INT-777

LCA

0

25000

50000

75000

100000

Rel

ativ

e Fl

uore

scen

ce In

tens

ity

(UC

P1

/ DA

PI) ****

D FE

0

10000

20000

30000

Rel

ativ

e Fl

uore

scen

t Int

ensi

ty(T

OM

M20

/ D

AP

I) *

c

Tgr5 +/+ Tgr5 -/-

DMSO INT-777

TOMM20DAPI

TOMM20DAPI

TOMM20DAPI

TOMM20DAPI

DMSO INT-777Tgr5 +/+ Tgr5 +/+ + INT-777Tgr5 -/- Tgr5 -/- + INT-777

Supplementary Fig. 6. TGR5 activation induces mitochondrial fission. (A) Quantitative densitometry of the Western blots showed in Figure 6e (n = 6 per group). (B) Quantitative densitometry of the Western blots showed in Figure 6f (n = 6 pergroup). Results represent mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 vs.Tgr5+/+ group by one-way ANOVA followed by Bonferroni post-hoc test.

B0.00.51.01.52.02.5

pDR

P1S6

37/D

RP1

0.00.20.40.60.81.0

MFF

/PAR

P1

A

0.0

0.2

0.4

0.6

0.8

pDR

P1S6

37/D

RP1

Tgr5 +/+ Tgr5 +/+ + INT-777Tgr5 -/- Tgr5 -/- + INT-777

0.0

0.2

0.4

0.6

0.8pC

REB

/CR

EB *

0.00.20.40.60.81.0

MFF

/VD

AC1

*

0

1

2

3

4

pDR

P1S6

16/D

RP1 *

0.0

0.2

0.4

0.6

pER

K/ER

K

*

0.0

0.5

1.0

1.5

2.0

pDR

P1S6

16/D

RP1 *

Supplementary Fig. 7. Effect of TGR5 activation on downstream signaling components during beige differentiation (A, C, E and G) Representative (n = 6 per group) Western blots of mitochondrial protein VDAC1 and TGR5 downstream targets (phospho proteins) with their relative controls (ERK, DRP1 or CREB) from differentiated adipocytes (different time points indicated in the figure) derived from the stromal vascular fraction (SVF) of TGR5 wild-type (Tgr5+/+) and germline TGR5 knock-out (Tgr5-/-

) mice. PARP1 was used as a loading control. Cells were stimulated with the TGR5 agonist INT-777 or vehicle (DMSO). n = 6 (B) Quantitative densitometry of the Western blots showed in A. (D) Quantitative densitometry of the Western blots showed in C. (F) Quantitative densitometry of the Western blots showed in E. (H) Quantitative densitometry of the Western blots showed in G. Results represent mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 vs. Tgr5+/+ DMSO group by one-way ANOVA followed by Bonferroni post-hoc test.

A C

E G

0.0

0.5

1.0

1.5

2.0

VDAC

1/PA

RP1

B

D

0

1

2

3

VDAC

1/PA

RP1

0.0

0.5

1.0

1.5

pDR

P1S6

16/D

RP1

0.0

0.5

1.0

1.5

2.0

pDR

P1S6

16/D

RP1

0.0

0.5

1.0

1.5

pER

K/ER

K

F

H

0

1

2

3

VDAC

1/PA

RP1

0.00.51.01.52.02.5

VDAC

1/PA

RP1

0.0

0.5

1.0

1.5

2.0

pER

K/ER

K *

0.0

0.5

1.0

1.5

2.0pD

RP1

S616

/DR

P1*

0.0

0.5

1.0

1.5

pDR

P1S6

16/D

RP1 ***

0.0

0.1

0.2

0.3

0.4

pER

K/ER

K

**

0.0

0.5

1.0

1.5

pER

K/ER

K

**

Tgr5 +/+ Tgr5 +/+ + INT-777Tgr5 -/- Tgr5 -/- + INT-777

+−

pERK

ERKpDRP1S616

DRP1VDAC1

INT-777DMSO+ + + − − − −

− − − + + ++

Tgr5+/+

Tgr5-/-

Tgr5+/+

Tgr5-/-

1d Diff.

PARP1

pCREBCREB

+−

pERK

ERK

pDRP1S616

DRP1VDAC1

INT-777DMSO+ + + − − − −

− − − + + ++

Tgr5+/+

Tgr5-/-

Tgr5+/+

Tgr5-/-

PARP1

3d Diff.

pCREBCREB

75

75

37

50

50

37

150

10050

50

+−

pERK

ERK

pDRP1S616

DRP1VDAC1

INT-777DMSO+ + + − − − −

− − − + + ++

Tgr5+/+

Tgr5-/-

Tgr5+/+

Tgr5-/-

PARP1

5d Diff.

pCREBCREB

+−

pERK

ERK

pDRP1S616

DRP1VDAC1

INT-777DMSO+ + + − − − −

− − − + + ++

Tgr5+/+

Tgr5-/-

Tgr5+/+

Tgr5-/-

PARP1

7d Diff.

pCREBCREB

75

75

37

50

50

37

150100

50

50

*

0.0

0.5

1.0

1.5

pCR

EB/C

REB

*

0.0

0.5

1.0

1.5

pCR

EB/C

REB

0.0

0.5

1.0

1.5

pCR

EB/C

REB

**

0.00

0.25

0.50

0.75

1.00

pCR

EB/C

REB

75

75

37

50

50

37

150

100

50

50

75

75

37

50

50

37

150

100

50

50

Supplementary Fig. 8. ERK activation is required for the TGR5-mediated mitochondrial fission. (A) Quantitative densitometry of the Western blots showed in Figure 7, a and b (n = 6 per group). (B) Quantification of mitochondrial (16S) versus nuclear (HK2) DNA ratio from differentiated adipocytes derived from the stromal vascular fraction (SVF) of TGR5 wild-type (Tgr5+/+) and germline TGR5 knock-out (Tgr5-/-) mice, differentiated in the presence or absence of the TGR5 agonist INT-777 and/or the ERK inhibitor FR180204. (C) Spare respiratory capacity calculated as the difference between maximal (FCCP-driven) and basal respiration measured in Figure 7d (n = 6 per group). Results represent mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 vs. Tgr5+/+ cells by one-way ANOVA followed by Bonferroni post-hoc test.

A

B

Tgr5 -/-

0.0

0.5

1.0

1.5

2.0

TBX1

/PAR

P1

0.0

0.5

1.0

1.5

2.0

VDAC

1/PA

RP1

0.0

0.2

0.4

0.6

0.8

pDR

P1S6

37/D

RP1

Tgr5+/+ DMSO Tgr5+/+ DMSO + FR180204 Tgr5+/+ INT-777 + FR180204Tgr5+/+ INT-777

0.0

0.3

0.6

0.9

1.2

PGC

-1α

/PAR

P1

0.0

0.5

1.0

1.5

2.0

TBX1

/PAR

P1

0.0

0.3

0.6

0.9

1.2U

CP1

/PAR

P1

0.0

0.5

1.0

1.5

2.0

VDAC

1/PA

RP1

0.0

0.5

1.0

1.5

2.0

pCR

EB/C

REB

0.0

0.3

0.6

0.9

1.2

pER

K/ER

K

0.00.20.40.60.81.0

pDR

P1S6

16/D

RP1

0.0

0.2

0.4

0.6

0.8

pDR

P1S6

37/D

RP1

0.0

0.3

0.6

0.9

1.2

UC

P1/P

ARP1 *

0.00.20.40.60.81.0

pDR

P1S6

16/D

RP1 **

0.0

0.5

1.0

1.5

2.0

pCR

EB/C

REB

****

0.0

0.3

0.6

0.9

1.2

pER

K/ER

K

**

C

0

100

200

300

Spar

e re

spia

rtory

cap

acity

(F

CC

P-Ba

sal O

CR

)Tgr5 -/-

0

100

200

300

Spar

e re

spia

rtory

cap

acity

(F

CC

P-Ba

sal O

CR

)

Tgr5 +/+

*

Tgr5 +/+

*

0.00.51.01.52.02.5

16S/

HK2

*

0.00.51.01.52.02.5

16S/

HK2

0.0

0.3

0.6

0.9

1.2

PGC

-1α

/PAR

P1

****

Tgr5-/- DMSO Tgr5-/- DMSO + FR180204Tgr5-/- INT-777 Tgr5-/- INT-777 + FR180204

Supplementary Fig. 9. Uncropped scans of Western blots related to Figure 1. Scans of (A) peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a), transcription factor T-box 1 (TBX1) and uncoupling protein 1 (UCP1), (B) voltage dependent anion channel 1 (VDAC1), and (C) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Western blots related to Figure 1f. Images represent three of more technical experiments.

VDAC1

GAPDH

100 kDa

50 kDa

75 kDa

37 kDa

25 kDa

25 kDa

37 kDa50 kDa

37 kDa

25 kDa

A

B

C

PGC1a

TBX1

UCP1

Supplementary Fig. 10. Uncropped scans of Western blots related to Figure 2. Scans of (A) peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a), (B) transcription factor T-box 1 (TBX1), (C) uncoupling protein 1 (UCP1), (D) voltagedependent anion channel 1 (VDAC1), and (E) glyceraldehyde 3-phosphatedehydrogenase (GAPDH) Western blots related to Figure 2f. Images represent three ofmore technical experiments.

150 kDa

50 kDa

75 kDa

37 kDa

25 kDa

37 kDa

25 kDa

37 kDa

50 kDa

A

B

C

D

E

37 kDa

100 kDaPGC1a

TBX1

UCP1

VDAC1

GAPDH

Supplementary Fig. 11. Uncropped scans of Western blots related to Figure 3. Scans of (A) peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a), (B) transcription factor T-box 1 (TBX1), (C) voltage dependent anion channel 1 (VDAC1)and uncoupling protein 1 (UCP1), and (D) glyceraldehyde 3-phosphate dehydrogenase(GAPDH) Western blots related to Figure 3e. Images represent three of more technicalexperiments.

TBX1

VDAC1

UCP1

GAPDH

100 kDa75 kDa

37 kDa

50 kDa

37 kDa

25 kDa

150 kDa

37 kDa

37 kDa

25 kDa

25 kDa

150 kDa

100 kDa75 kDa

A

B

C

D

PGC1a

Supplementary Fig. 12. Uncropped scans of Western blots related to Figure 4. Scans of (A) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a) and voltage dependent anion channel 1 (VDAC1), (B) transcription factor T-box 1 (TBX1) and uncoupling protein 1 (UCP1) Western blots related to Figure 4c. Images represent three of more technical experiments.

25 kDa

25 kDa

37 kDa

37 kDa

50 kDa

37 kDa

37 kDa

25 kDa

100 kDaPGC1a

GAPDH

TBX1

VDAC1

UCP1

A

B

75 kDa

Supplementary Fig. 13. Uncropped scans of Western blots related to Figures 5 and 6. Scans of (A) peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a), (B) poly(ADP-ribose) polymerase 1 (PARP1), (C) transcription factor T-box 1 (TBX1) and uncoupling protein 1 (UCP1), (D) phosphorylation of cyclic AMP response element binding protein (pCREB) and phosphorylation of extracellular signal-regulated kinase (pERK), and (E) cyclic AMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK) Western blots related to Figures 5d and 6e. Images represent three of more technical experiments.

100 kDa150 kDa

75 kDa

37 kDa

37 kDa

37 kDa

37 kDa

50 kDa

50 kDa

50 kDa

50 kDa

50 kDa

100 kDa150 kDa

PGC1a

PARP1

TBX1

pCREB

UCP1

CREB

pERK

ERK

A

B

C

D

E

Supplementary Fig. 14. Uncropped scans of Western blots related to Figure 6. Scans of (A) translocase of outer mitochondrial membrane 40 (TOMM40), (B) phosphorylation in serine 637 of dynamin-1-like protein (pDRP1S637) and phosphorylation in serine 616 of dynamin-1-like protein (pDRP1S616), (C) dynamin-1-like protein (DRP1), (D) mitochondrial fission factor (MFF), and (E) poly(ADP-ribose) polymerase 1 (PARP1)Western blots related to Figures 5d and 6e. Images represent three of more technicalexperiments.

37 kDa

100 kDa

75 kDa

75 kDa

50 kDa

75 kDa

50 kDa

37 kDa

25 kDa

50 kDa

150 kDaPARP1

MFF

DRP1

TOMM40

E

D

B

C

A

pDRP1S637

pDRP1S616

Supplementary Fig. 15. Uncropped scans of Western blots related to Figure 7 part 1. Scans of (A) peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC1a),(B) transcription factor T-box 1 (TBX1), (C) uncoupling protein 1 (UCP1), (D) voltagedependent anion channel 1 (VDAC1) and (E) phosphorylation of cyclic AMP responseelement binding protein (pCREB) and cyclic AMP response element binding protein(CREB) Western blots related to Figures 7a and b. Images represent three of moretechnical experiments.

PGC1a

TBX1

UCP1

VDAC1

pCREB

CREB37 kDa

50 kDa

50 kDa

37 kDa

50 kDa

50 kDa

37 kDa

50 kDa

37 kDa

25 kDa

75 kDa

50 kDa

100 kDa75 kDa

100 kDa

50 kDa

37 kDa

25 kDa

37 kDa

50 kDa

A

B

C

D

E

Supplementary Fig. 16. Uncropped scans of Western blots related to Figure 7 part 2. Scans of (A) phosphorylation of extracellular signal-regulated kinase (pERK), (B)phosphorylation in serine 616 of dynamin-1-like protein (pDRP1S616), (C) phosphorylationin serine 637 of dynamin-1-like protein (pDRP1S637), (D) extracellular signal-regulatedkinase (ERK) and (E) poly(ADP-ribose) polymerase 1 (PARP1) Western blots related toFigures 7a and b. Images represent three of more technical experiments.

37 kDa

50 kDa

75 kDa100 kDa

37 kDa50 kDa

75 kDa100 kDa

75 kDa100 kDa75 kDa

100 kDa

37 kDa50 kDa

37 kDa50 kDa

pERK

ERK

PARP1

pDRP1S637

pDRP1S616

A

B

C

D

E150 kDa100 kDa75 kDa

100 kDa150 kDa

75 kDa

Supplementary Table 1. Primers used for qPCR

Specie Gene Sequence 5’ – 3’ Mouse Pgc1a Fw TGAGGACCGCTAGCAAGTTT

Rv TGTAGCGACCAATCGGAAAT Ucp1 Fw CTTTGCCTCACTCAGGATTGG

Rv ACTGCCACACCTCCAGTCATT Tbx1 Fw CTGTGGGACGAGTTCAATCAG

Rv TTGTCATCTACGGGCACAAAG Prdm16 Fw CAGCACGGTGAAGCCATTC

Rv GCGTGCATCCGCTTGTG Cidea Fw CATGATCTTGGAAAAGGGACAG

Rv ATCGTGGCTTTGACATTGAGAC Cd137 Fw CCTTGCAGGTCCTTACCTTGT

Rv GTTGCTTGAATATGTGGGGGA Pparg2 Fw TCGCTGATGCACTGCCTATG

Rv GAGAGGTCCACAGAGCTGATT Cebpb Fw GCAAGAGCCGCGACAAG

Rv GGCTCGGGCAGCTGCTT Adrb3 Fw ACAGCAGACAGGGACAGAGG

Rv TCCTGTCTTGACACTCCCTCA Th Fw GTCTCAGAGCAGGATACCAAGC

Rv CTCTCCTCGAATACCACAGCC Tgr5 Fw TCCTGTCAGTCTTGGCCTATGA

Rv GGTGCTGCCCAATGAGATG 36b4 Fw AGATTCGGGATATGCTGTTGG

Rv AAAGCCTGGAAGAAGGAGGTC B2m Fw CTGACGAAGGCCACTTTGCACCTCTG

Rv GCACAAGATCCCGGTCAAGTACCTGGAG 16S Fw CCGCAAGGGAAAGATGAAAGAC

Rv TCGTTTGGTTTCGGGGTTTC Hk2 Fw GCCAGCCTCTCCTGATTTTAGTGT

Rv GGGAACACAAAAGACCTCTTCTGG Human Pgc1a Fw TCTGAGTCTGTATGGAGTGACAT

Rv CCAAGTCGTTCACATCTAGTTCA Ucp1 Fw AGGTCCAAGGTGAATGCCC

Rv GCGGTGATTGTTCCCAGGA Tbx1 Fw TAGCGAGAAATATGCCGAGGA

Rv CGTGATCCGATGGTTCTGGT Prdm16 Fw CGAGGCCCCTGTCTACATTC

Rv GCTCCCATCCGAAGTCTGTC Cidea Fw TTATGGGATCACAGACTAAGCGA

Rv TGCTCCTGTCATGGTTGGAGA Cd137 Fw AGCTGTTACAACATAGTAGCCAC

Rv TCCTGCAATGATCTTGTCCTCT Pparg2 Fw CAGCAAACCCCTATTCCATGC

Rv TCTCCGGAAGAAACCCTTGC Cebpb Fw AGAGCAAGGCCAAGAAGACC

Rv GACAGCTGCTCCACCTTCTT Hprt Fw TGGGATTACACGTGTGAACCAACC

Rv GCTCTACCCTCTCCTCTACCGTCC