Chr 1Ctdsp1

1 kb

miR-26b

Chr 10Ctdsp2

2 kb

miR-26a-2

Ctdspl

miR-26a-1

10 kb

Chr 9

A

0

1

2

3

4

5

Heart

Lung

Kidney

S Musc

le

Colon

S Inte

stin

eLiv

er

Spleen

Lymph N

ode

Bone M

arro

w

Cereb

ellu

m

Cortex

Testis

Re

lative

Exp

ressio

n

miR-26a

B

0

1

2

Heart

Lung

Kidney

S Musc

le

Colon

S Inte

stin

eLiv

er

Spleen

Lymph N

ode

Bone M

arro

w

Cereb

ellu

m

Cortex

Testis

Re

lative

Exp

ressio

n

miR-26b

C

Supplemental Figure S1. miR-26 host transcripts and expression patterns of miR-26 family members in mice. (A) Schematic representation of miR-26 host transcripts. (B-C) qRT-PCR analysis of mature miR-

26a (B) and miR-26b (C) expression in tissues of 3 month old mice (n=4). Expression is shown relative to

liver. Data represented as mean ± s.d.

A

Genomic locus:

miR-26 sgRNA:

miR-26a-1 miR-26a-2 miR-26b

26a-126a-2

26b NT26a-1

26a-226b NT

26a-126a-2

26b NT

*

*

**

*

*

254

368

195

333

291

357

D

0

1

2

3

Heart

Liver

Lung

Sk Mus

WAT

BAT

Re

lative

Exp

ressio

n

miR-26a WT

TKO

E

0

0.5

1

1.5

Heart

Liver

Lung

Sk Mus

WAT

BATR

ela

tive

Exp

ressio

n

miR-26b WT

TKO

+/+ +/− −/−

miR-26a-1

miR-26a-2

miR-26b

D26

D20

D31

B

WT 5/6-KO TKOC

0

0.5

1

1.5

Ctdsp1 Ctdsp2 Ctdspl

Re

lative

exp

ressio

n

Sk Muscle WTTKO

0

0.5

1

1.5

Ctdsp1 Ctdsp2 Ctdspl

Re

lative

exp

ressio

n

Liver WTTKO

0

0.5

1

1.5

Ctdsp1 Ctdsp2 Ctdspl

Re

lative

exp

ressio

n

WAT WT

TKO

F G H

Supplemental Figure S2. Generation and analysis of miR-26-TKO mice. (A) Cas9-induced indels at miR-26a-1, miR-26a-2, and miR-26b genomic loci in mouse ES cells transfected with Cas9 and sgRNAs targeting each miRNA, detected by T7 endonuclease assay. Expected sizes (number of base pairs) of cleavage products (denoted by *) are indicated below the image. (B) Representative PCR-based genotyping assay of miR-26a-1, miR-26a-2, and miR-26b knockout alleles. (C) Gross appearance of 3 month old mice of the indicated genotypes. (D-E) qRT-PCR analysis showing undetectable expression of mature miR-26a (D) and miR-26b (E) in tissues of TKO mice. (F-H) qRT-PCR analysis of miR-26 host gene expression in the indicated tissues. Data represented as mean ± s.d.

A

0

0.5

1

1.5

2

B.W IGW

ISCW

BATLive

r

Kidney

Spleen

Heart

Rel

ativ

e w

eigh

t

WT5/6-KOTKO

E

0

50

100

150

Wt

5/6-K

OTKO

Cho

lest

erol

(mg/

dL)

D

0

100

200

300

400

Wt

5/6-K

OTKO

Trig

lyce

rides

(mg/

dL)

C

0

5

10

15

20

25

WT

5/6-K

OTKO

% B

ody

Fat

B

0

1

2

3

4

IGW

ISCW

BATPGW

RPWMW

ATLive

r

Kidney

Spleen

Pancr

eas

Sk Musc

leHea

rt

Rel

ativ

e W

eigh

t

WT5/6-KOTKO

******

Fat depots

**

F

0

10

20

30

WT

5/6-K

OTKO

B.W

. (gm

s)

0

5

10

15

20

25

WT

5/6-K

OTKO

B.W

. (gm

s)

G

0

50

100

150

WT

5/6-K

OTKO

Bloo

d G

lc (m

g/dL

)

H

0

50

100

150

WT

5/6-K

OTKO

Bloo

d G

lc (m

g/dL

)

J K

0

50

100

150

200

250

300

350

400

0 15 30 45 60 90 120

Glu

cose

(mg/

dL)

Time (min)

GTT

WTTKO

0

50

100

150

200

250

300

0 15 30 45 60 90 120

Glu

cose

(mg/

dL)

Time (min)

GTTWTTKO

** **

**

** **

* ******

**

WT

WT

I

Supplemental Figure S3. miR-26-TKO mice have excess body fat but normal glucose tolerance. (A) Relative weights of isolated fat depots and major organs from 3 month old female mice of the indicated genotypes fed standard chow, normalized to wild-type (n=6 mice per genotype). Fat depots: IGW: iguinal; ISCW: interscapular; PGW: perigonadal; RPW: retroperitoneal; MWAT: mesenteric; BAT: brown adipose tissue. (B) Relative weights of fat depots and major organs isolated from postnatal day 10 female mice (n=4-6 mice per genotype). (C) Fat content of 3 month old female mice measured by NMR (n=6 per genotype). (D-E) Serum triglycerides (D) and cholesterol (E) in 3 month old female mice (n=6 per genotype). (F-G) Blood glucose levels of 3 month old male (F) and female (G) mice (n=6-8 per genotype). (H-I) Glucose tolerance test (GTT) performed on 8 week old male (H) and female (I) mice (n=6 per genotype). (J-K) Total body weight of 3 month old male (J) and female (K) mice (n=6-8 per genotype). Data represented as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001, calculated using two-tailed t test.

Adult (♀)

Adult (♀) Adult (♀) Adult (♀) Adult (♀)Adult (♂)

Adult (♀)Adult (♂) Adult (♀)Adult (♂)

Developmental (♀)

A

0

1

2

3

Light Dark

Food

Inta

ke (g

ms/

day)

WT5/6-KOTKO

0

1

2

3

4

5

6

Light Dark

VO

2(L

/Kg/

hr)

WT5/6-KOTKO

0

1

2

3

4

5

Light Dark

VC

O2

(L/k

g/hr

)

WT5/6-KOTKO

B

C D

0

1

2

3

Light Dark

Phy

sica

l act

ivity

(x10

3be

am b

reak

s)

WT5/6-KOTKO

Supplemental Figure S4. miR-26-TKO mice do not exhibit altered food intake or energy expenditure. (A-D) Mice (n=4 per genotype) were monitored over a 5 day period in metabolic chambers to quantify average food intake per 12 hour light and dark cycle (A); physical activity as measured by average beam breaks along X, Y and Z axes per light/dark cycle (B); average oxygen consumption per hour per light/dark cycle (C); and average carbon dioxide production per hour per light/dark cycle (D). Data represented as mean ± s.d. No significant differences were noted using two-tailed t test.

0

0.5

1

1.5

IGW

ISCW

BAT

PG

W

RPW

MW

AT

Liv

er

Kid

ney

Sple

en

Pancre

as

Sk M

uscle

Heart

Rel

ativ

e W

eigh

t

CtrlM2rtTA-26a

M2rt

TA

M2rt

TA

;

26aM

2rtTA

M2rt

TA

;

26a

A

0

10

20

30

40

50

60

Ctrl

M2rtTA-2

6a

B.W

. (gm

s)

B

0

10

20

30

40

50

Ctrl

M2rtTA-2

6a

% B

ody

Fat

C D

E

**** **

**** **

*

**

**

Supplemental Figure S5. Weight gain on normal chow in mice that globally overexpress miR-26a. (A) Representative images of M2rtTA and M2rtTA;miR-26a transgenic mice maintained on dox and normal chow for 1 year. Dox was administered starting at 6 weeks of age. (B) Total body weight of male mice of the indicated genotypes maintained on dox and normal chow for 1 year. Control (Ctrl) mice represent M2rtTA or miR-26a single transgenic animals (n=6 per genotype). (C) Body fat content of mice in (B), measured by whole body NMR. (D) Representative images of isolated fat depots from mice of the indicated genotypes maintained on dox and normal chow for 1 year. (E) Relative weight of fat depots and major organs from mice in (B). Data represented as mean ± s.d. *p<0.01, **p<0.001, calculated using two-tailed t test.

C

0

0.5

1

1.5

miR

-26a

miR

-26b

Rel

ativ

e Ex

pres

sion

WT

TKO

0

1

2

3

4

Adip

sin

aP2

Adpn

Cebpa

FASN

Glu

t4

Leptin

LPL

Pparg

2

Retn

Sre

bp1c

Rel

ativ

e Ex

pres

sion

WT

TKO

F G

WT

TK

O

H

E

0

1

2

3

Adip

sin

aP2

APN

CEBPa

FASN

Glu

t4

Leptin

LPL

Pparg

2

Resis

tin

SREBP1c

Rel

ativ

e Ex

pres

sion

−Dox

+Dox

M2rtTA

−D

ox

+D

ox

0

0.5

1

1.5

miR

-26a

miR

-19b

Rel

ativ

e Ex

pres

sion

−Dox

+Dox

Supplemental Figure S6: miR-26 regulates adipogenesis in vitro. (A) Schematic of transgenes in Adipotrak mice (Pparg-tTA; TRE-H2B-GFP) used for GFP-labeling of adult APCs. (B) qRT-PCR analysis of mature miR-26a and miR-26b in FACS-sorted GFP+ cells from SVF isolated from pooled fat depots from five P30 Adipotrak mice (n=3 technical replicates). Data is represented relative to expression in GFP− population. (C) qRT-PCR analysis of miR-26a and miR-26b levels in SVFs derived from wild-type and miR-26-TKO mice at 5 weeks of age. n=3 biological replicates per condition. (D) qRT-PCR analysis of adipogenic gene expression following in vitro differentiation of SVF cultures from (C). (E) Representative images of Oil Red O-stained SVF cultures from (C). (F) qRT-PCR analysis of mature miR-26a and miR-26b expression in SVF cultures derived from M2rtTA transgenic mice, cultured with or without dox (1 µg/ml) for 4 days prior to induction of differentiation. n=3 biological replicates per condition. (G) qRT-PCR analysis of adipogenic gene expression following in vitro differentiation of dox-treated SVF cultures from (F), relative to untreated cultures.(H) Representative images of Oil Red O-stained SVF cultures from (F). Data represented as mean ± s.d.*p<0.05, **p<0.01, calculated using two-tailed t test.

* **

***

** ***

****

0

0.5

1

1.5

2

miR

-26a

miR

-26b

Rel

ativ

e Ex

pres

sion

GFP−GFP+

A

TRE H2B-GFP

tTA

Pparg-tTA

TRE-H2B-GFP

B

Adipotrak

D

Wild-type miR-26-TKO

R26RtdT/+

A BWild-type

tdTo

mat

o+

Sca-1+

2.2%miR-26-TKO

tdTo

mat

o+

Sca-1+

1.7%

C D

0

0.5

1

1.5

−Dox +Dox

Rel

ativ

e S

tradB

expr

essi

on

0

0.5

1

1.5

2

2.5

WT TKO

Rel

ativ

e S

tradB

expr

essi

on

**

**

Supplemental Figure S7. Analysis of APC population and miR-26 targets in adipogenesis. (A) Whole mount images of SMA-CreERT2/+; R26RtdT/+ labeled IGW fat depots from mice of the indicated genotypes, treated with tamoxifen at P25 and analyzed at P50. Arrows and arrowheads indicate labeled vasculature and mature adipocytes, respectively. (B) FACS analysis of Sca1+ and tdTomato+ positive APCs isolated from control (SMA-CreERT2/+; R26RtdT/+) or miR-26-TKO (miR-26-TKO; SMA-CreERT2/+; R26RtdT/+) mice treated with tamoxifen at 5 weeks of age and analyzed 24 hours later. Gates were set using unstained and singly-stained (tdTomato or Sca-1 only) samples (data not shown). (C-D) qRT-PCR analysis of StradB expression in SVF cultures from wild-type or miR-26-TKO mice (C) or miR-26a transgenic mice with or without dox treatment (D). (E) Relative expression of the indicated genes, assessed by qRT-PCR, 48 hours after transfection of wild-type SVF cultures with the indicated siRNAs. siNT, non-targeting control siRNA. (F) Representative images of Oil Red O-stained cultures from (E) following in vitro differentiation. (G) qRT-PCR analysis of adipogenic gene expression in cultures from (E) following in vitro differentiation. Data represented as mean ±s.d. **p<0.01, calculated using two-tailed t test.

G

siNT

siStradb

siFbxl19

siPparg2

E

0

0.5

1

1.5

2

Fbxl19 StradB Pparg2

Rel

ativ

e Ex

pres

sion

siNTsiFbxl19siStradBsiPparg2

siNTsiFbxl19siStradBsiPparg2

F

0

1

2

3

Adipsin

Fabp4

Adpn

Cebpa

Fasn

Glut4

Leptin Lpl

Pparg1

Pparg2

Retn

Srebp1c

Rel

ativ

e ex

pres

sion

siNTsiFbxl19siStradB