Sup

Sup

(A)

1-m

qPC

MS

CD3

by F

pplemental

pplemental

Co-localiz

month-old b

CR detectio

C FACS em

31‐Ter119

FACS in BM

figures and

Figure S1.

zation of F

one marrow

on of Foxp1

mploying e

‐CD29+Sca

M from 1 an

d Figure le

. Expressio

Foxp1 and

w (BM). (B

1A, B, C, a

expression (

a1+. (D) qP

nd 18- mont

1 

egends

n of Foxp1

d Nestin in

B) Location

and D isofo

(+) or abse

PCR detectio

ths-old dono

1 during M

n cultures

ns of oligon

forms. (C) R

ence (-) of

on of Foxp

ors.

SC differen

of MSCs

nucleotide p

Representat

standard m

1 and p16 i

ntiation.

isolated f

probes used

tive dot plo

markers: CD

in MSCs so

from

d for

ot of

D45‐

orted

 

Sup

(A,

qPC

loss

and

The

trab

of t

BV/

bon

Cb.T

pplemental

B) Assessm

CR and west

s of volume

BMD of co

e volume,

becular sepa

trabecular b

/TV, bone v

ne number;

Th., cortica

Figure S2.

ment of Fox

tern blotting

e, number, a

ortical bone

number an

aration was

bones and p

volume/tiss

Tb.Sp., trab

al bone thick

. Bone prop

xp1 express

g. **, P<0.0

and BMD o

es in Foxp1P

nd BMD o

increased in

parameters o

ue volume;

becular bon

kness. *indi

2 

perties of F

sion in BM

01; n=4. (C

of trabecula

Prx1Δ/Δ mutan

of trabecul

n mutants a

of cortical b

; BMD, bon

ne separatio

icates P<0.0

Foxp1Prx1Δ/Δ

M MSCs from

C, D) μCT an

ar bones as

nts between

lar bones

as compared

bones were

ne mineral

on; Tb.Th.,

05; **, P<0

female mu

m Foxp1Prx

nalyses dete

well as volu

n 6 and 16 m

were decre

d to controls

e unaltered.

density; Tb

trabecular b

.01; ***, P<

utants.

x1Δ/Δ mutant

ects progres

lume, thickn

months. n=4

eased, whe

s. The thick

Abbreviati

b.N., trabec

bone thickn

<0.001; n=4

ts by

ssive

ness,

4. (E)

ereas

kness

ions:

cular

ness;

4.

 

Sup

(A)

(B)

oste

as

CD3

num

pplemental

TRAP stain

Quantifica

eoclast prog

defined b

3-B220-CD

mbers in (C)

Figure S3.

ning for trab

ation of oste

genitor cells

by FACS

11b-CSF1R

); ns, nonsig

. TRAP stai

becular bon

eoclast num

s in BM Fo

expression

R+cKit+ . (D

gnificant; n=

3 

ining for os

nes of Foxp

mbers in (A

oxp1 mutant

(+) or

D) Quantifi

=3.

steoclasts i

1 mutant an

A). (C) Dot

t and contro

absence (-

fication of

n Foxp1Prx1

nd control m

plot of FA

ol mice at 3

-) of stan

osteoclast

1Δ/Δ mice.

mice at 3 of

ACS analysi

3 months of

ndard mark

progenitor

f age.

is of

f age

kers:

cell

 

Sup

oste

(A)

(pM

cult

adip

ALP

anal

MS

RT-

Sup

mic

pplemental

eoblasts in

Foxp1 w

MSCV-Foxp

tured in dif

pogenic ind

P staining. (

lyzed by R

Cs cells. (

qPCR follo

pplemental

ce.

Figure S4

MSCs.

was overex

1) followed

fferentiation

duction by o

(B) The exp

RT-qPCR 6

(C) Expres

owing 14 da

Figure S5.

4. Foxp1 re

pressed in

d by stable

n medium.

oil red O st

pression of a

days follow

sion of bo

ys of osteog

. Histologic

4 

egulates th

n murine M

e-transfectio

Cell differ

taining or 1

adipogenic

wing adipo

one markers

genic cultur

cal analysis

he different

MSCs by

on of MSC

rentiation w

14 days afte

markers (C

genic induc

s (Alp and

re. *, P<0.0

s of the bon

tiation of a

insertion

Cs and the

was assesse

er osteogen

CEBP, PPA

ction of Fo

d Col1a1) a

5; ***, P<0

ne marrow

adipocytes

into retrov

en selected

ed 6 days

nic induction

AR and Fa

oxp1-expres

as assessed

0.001; n=3.

w in Foxp1N

and

virus

and

after

n by

bp4)

ssing

d by

NesΔ/Δ

5  

(A) Growth curve of Foxp1NesΔ/Δ mice from measured at days after passage (P) from

P3 to P21. (B) H&E staining of tibias from Foxp1fl/fl and Foxp1NesΔ/Δ mice at P21.

Representative images were taken from the primary ossification region below the

growth plates. (C) Oil red O staining of the tibias of (B) at P21. Representative

images were taken from the secondary ossification proximal to the knee joint. (D)

Quantification of the adipose droplets in (C); ***, P<0.001; n=5.

 

Sup

in F

(A)

pplemental

Foxp1NesΔ/Δ

Represen

Figure S6.

mice.

ntative ima

. The altere

ages of F

6 

ed osteogen

Foxp1NesΔ/Δ

nic and adip

and Foxp

pogenic po

p1fl/fl mice

otency of M

at P25.

MSCs

(B)

 

Rep

Foxp

BV/

rela

decr

P<0

asse

Oste

stain

adip

***

BM

Sup

(A)

mut

dete

BV/

bon

presentative

xp1NesΔ/Δ at

/TV, BMD

atively incre

reased but c

0.001; n=5.

essed 14 da

eogenic dif

ning at 21 d

pocyte colo

, P<0.001;

M mesenchym

pplemental

Representa

tant and Fo

ect no chan

/TV, bone v

ne number; T

images of

P21. (C) Q

D, Tb.N, T

eased. Simi

cortical BM

(D) The a

ays after a

fferentiation

days of diff

onies (CFU-

n=4. (G) W

mal progeni

Figure S7.

ative image

oxp1fl/fl cont

nges in bon

volume/tissu

Tb. Sp., trab

μCT analy

Quantificati

Tb.Th and

ilarly, Cb.T

MD was not

adipogenic

dipogenic i

n of mesenc

ferentiation

-Ad) and o

Western blot

itors from F

. Bone phen

s of trabecu

trol 3 month

ne propertie

ue volume;

becular bon7 

ysis for trab

ion of bone

Tb.Sp we

Th and CV/

significantl

potency of

induction b

chymal prog

induction (

steoblast co

identificati

Foxp1NesΔ/Δ

notypes in

ular and cor

hs old mice

es in Foxp

BMD, bon

ne separatio

becular and

e properties

re decrease

/TV of the

ly altered. *

f BM mese

by oil red

genitors wa

lower panel

olonies (CF

on of PPAR

and Foxp1f

the Foxp1C

rtical bones

e by μCT a

p1Col2Δ/Δ mu

ne mineral

on; Tb. Th.,

cortical bo

indicating

ed, wherea

cortical bo

, P<0.05; *

enchymal p

O staining

as assessed

l). (E, F) Q

FU-Ob) in (

Rγ and Fabp

fl/fl mice at P

Col2Δ/Δ mice.

from tibia

nalyses. (B

utant mice.

density; Tb

trabecular

ones of tibia

that trabec

as BS/BV

ones were

**, P<0.01;

progenitors

(upper pa

by Alizarin

Quantificatio

(D). *, P<0

p4 expressio

P21.

.

of Foxp1Co

B) μCT anal

Abbreviati

b. N., trabec

bone thickn

as in

cular

was

also

***,

was

nel).

n red

on of

0.05;

on in

ol2Δ/Δ

lyses

ions:

cular

ness;

 

Cb.

Safr

at 8

mes

Sup

CEB

(A)

P<0

RBP

RBP

Th., cortic

ranin O stai

8 months of

senchymal p

pplemental

BP.

Foxp1 rep

0.01; ***, P

Pjκ in the n

Pjκ-Flag ex

cal bone th

ining of the

f age. (D) A

progenitor c

Figure S8

pressed the

P<0.001; n=

nuclei of C3

xpression v

hickness. *,

e growth pla

Alcian blue

cells after 14

8. Foxp1 re

transactiva

=3. (B) Co

H10T1/2 ce

vectors. G8 

, P<0.05; *

ate of Foxp

e staining fo

4 days indu

epresses th

ation of PPA

o-localizatio

ells transfec

Green, anti-

**, P<0.01

p1Prx1Δ/Δ mu

or the chon

uction.

he transacti

PAR-Luc by

on of Foxp1

cted with Fo

Flag; red,

; ***, P<0

utant and Fo

drogenic di

ivation of P

y CEBP.

1 with CEB

oxp1 and CE

anti-Foxp1

0.001; n=5.

oxp1fl/fl con

ifferentiatio

PPAR-Luc

*, P<0.05;

BPβ, CEBP

EBPβ/δ-Fla

1; blue, D

(C)

ntrols

on of

c by

; **,

Pδ or

ag or

DAPI

9  

staining to identify nuclei. Bar, 50 μm. (C) Schematic diagram showing that the

mutation induced within the p16 promoter, which was employed in Figure 6C. (D)

Representative histograms of DCFDA mean fluorescence intensities in mesenchymal

progenitors of the second passages of cultures. (E) Expression of FOXP1 in P1, P3

and P5 passages of 27- and 74-year-old hMPCs following Foxp1 lentiviral-mediated

overexpression.

Oligos sequence for PCR

Primers for realtime PCR Species Name Sequence

Mouse

β-actin-F AGAGGGAAATCGTGCGTGACA β-actin-R CACTGTGTTGGCATAGAGGTC Foxp1-F TCTCGTCCTCGGCACCTT Foxp1-R GTCACAAACCGCCTCACA Nestin-F CACACCTCAAGATGTCCC Nestin-R GAAAGCCAAGAGAAGCCT Cebpα-F TGGACAAGAACAGCAACGAG Cebpα-R TCACTGGTCAACTCCAGCAC PPARγ-F GGAAAGACAACGGACAAATCAC PPARγ-R TACGGATCGAAACTGGCAC Fabp4-F GATGAAATCACCGCAGACGACA Fabp4-R ATTGTGGTCGACTTTCCATCCC p21-F GAACATCTCAGGGCCGAAAAC p21-R CTGCGCTTGGAGTGATAGAA p27-F ACTAACCCGGGACTTGGAGA P27-R GAAATTCCACTTGCGCTGAC p53-F GTCACAGCACATGACGGAGG p53-R TCTTCCAGATACTCGGGATAC p16-F CTAGAGAGGATCTTGAGAAGAGGGC p16-R TAGTTGAGCAGAAGAGCTGCTACGT Bmi-F CTACACGCTAATGGACATTGCCT Bmi-R CCATCCCTCTGGTGACTCATCTT Hey1-F CACTGCAGGAGGGAAAGGTTAT Hey1-R CCCCAAACTCCGATAGTCCAT HeyL-F GAAGCGCAGAGGGATCATAGA HeyL-R CCAATCGTCGCAATTCAGAA Jagged1-F CTTCAATCTCAAGGCCAGCC Jagged1-R CAGGCGAAACTGAAAGGCAG

10  

Alp-F GCCTGGATCTCATCAGTATTTGG Alp-R GTTCAGTGCGGTTCCAGACAT Col1α1-F CCGGAAGAATACGTATCACC Col1α1-R ACCAGGAGGACCAGGAAGTC Runx2-F CCGGGAATGATGAGAACTA Runx2-R ACCGTCCACTGTCACTTT Osterix-F CTCTCTGCTTGAGGAAGAAG Osterix-R GTCCATTGGTGCTTGAGAAG

Human

ALP-F AACATCAGGGACATTGACGTG ALP-R GTATCTCGGTTTGAAGCTCTTCC COL1A1-F GTGCGATGACGTGATCTGTGA COL1A1-R CGGTGGTTTCTTGGTCGGT HEY1-F ATCTGCTAAGCTAGAAAAAGCCG HEY1-R GTGCGCGTCAAAGTAACCT HEYL-F GGAAGAAACGCAGAGGGATCA HEYL-R CAAGCGTCGCAATTCAGAAAG ACTIN-F CCAGCACAATGAAGATCAAGAT ACTIN-R AGAAAGGGTGTAACGCAACTAA FOXP1-F GGGGCAGTATGGACAGTGGATGA FOXP1-R TTGAGAGGTGTGCAGTAGGCGTG

Primers for ChIP-PCR

Name Sequence Pparg-ChIP-F CCCATTGAGCTATTGCTTC Pparg-ChIP-R TCAGTGACTTGTGGACTTT p16-ChIP-F TACACAGTTATGAGTTAGGGCAAp16-ChIP-R CTTCTTGAGGTCTGTAAGGAAAA

Figure 1B

Foxp1

β-actin

1M 8M 30M

β-actin

Foxp1

26 27 33 41 74 75 82M

Figure 1E

Figure 2B

Foxp1

β-actin

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Figure 4F

PPARγ

β-actin

Fabp4

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Figure 4A Figure 4B

- + ++ - +

- + ++ - +

Cebpβ-Flag

Foxp1-His

Input Output

IP-His IB-Flag

IP-FlagIB-His

- + ++ - +

- + ++ - +

Cebpδ-Flag

Foxp1-His

Input Output

Figure 4C Figure 4J

Figure 4K

Foxp1

Cebpβ

Cebpδ

Rbpjk

- + ++ - +

- + ++ - +

Rbpjk-Flag

Foxp1-His

IP-HisIB-Flag

IP-FlagIB-His

Input IgG anti-Foxp1

Input Output

Foxp1

Figure 5G

Foxp1

LAP2ββ-actinH3K9me3

p16

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

β-actin

Foxp1 Pr

x1Δ/Δ

Foxp

1fl/fl

Figure 7H

FABP4

β-ACTIN

PPARγ

LV-G

FP

LV-FO

XP1

Foxp1 ne

sΔ/Δ

Foxp

1fl/fl

PPARγ

Fabp4

β-actin

Figure S6G