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SUPPLEMENTARY INFORMATION
Prdm4 induction by the small molecule butein promotes white adipose tissue browning
No-Joon Song1, Seri Choi2,#, Prashant Rajbhandari3,#, Seo-Hyuk Chang1, Suji Kim1, Laurent Vergnes4 So Mi Kwon1 Jung Hoon Yoon1 Suk Chan Lee5 Jin Mo Ku6 JeongLaurent Vergnes4, So-Mi Kwon1, Jung-Hoon Yoon1, Suk-Chan Lee5, Jin-Mo Ku6, Jeong-Soo Lee7, Karen Reue4,8, Seung-Hoi Koo2, Peter Tontonoz3,9, Kye Won Park1,*
1Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 16419, Korea.2Division of Life Sciences, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Korea.f f , y, , g , ,3 Howard Hughes Medical Institute and 9Departments of Pathology and Laboratory Medicine, University of California, Los Angeles, California 90095, USA.4Department of Human Genetics,8Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA. 5Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Korea.6Natural product research team, Gyeonggi Bio-Center, Gyeonggi Institute of Science and Technology promotion, Suwon 16229, Korea.7Korea Research Institute of Bioscience and Biotechnology and Major of Functional Genomics, University of Science f gy j f y fand Technology, Daejeon 34141, Korea.
#These authors contributed equallyThese authors contributed equallyCorresponding author : [email protected]
Nature Chemical Biology: doi:10.1038/nchembio.2081
SUPPLEMENTARY RESULTS
Supplementary Table 1: Ucp1 induction by bioactive compoundsSupplementary Table 1: Ucp1 induction by bioactive compounds.
Supplementary figure1. Identification of butein as an Ucp1 inducer.Supplementary figure 2: Butein inhibits adipogenesis in C3H10T1/2 cells.Supplementary figure 3: Butein induces expression of thermogenic genes in adipocytes.S l fi 4 S l i i d i f U 1 b b iSupplementary figure 4: Selective induction of Ucp1 by butein.Supplementary figure 5. Identification of genes selectively regulated by butein.Supplementary figure 6: Effects on Ucp1 expression by butein-selective genes.Supplementary figure 7: Induction of Prdm4 and Ucp1 by butein in vivo.Supplementary figure 8: Acute induction of Prdm4 by butein.pp y g ySupplementary figure 9: Prdm4 silencing increases lipid accumulation and expression of white adipocyte-selective genes in preadipocytes.Supplementary figure 10: Prdm4 regulates oxygen consumption rates in C3H10T1/2 cells.Supplementary figure 11: Prdm4 induces Ucp1 and thermogenic genes in T37i cells.Supplementary figure 12: Prdm4 overexpressing cells decrease expression of white adipocyte-selective genesSupplementary figure 12: Prdm4 overexpressing cells decrease expression of white adipocyte-selective genes. Supplementary figure 13: Prdm4 is necessary for the effects of butein in thermogenic programming. Supplementary figure 14: Prdm4 expression is reduced in adipose tissues of obese and diabetic mice.Supplementary figure 15: Effects of Prdm4 knockdown on tissue weight gains and insulin sensitivity in HFD fed mice.Supplementary figure 16: Prdm4 knockdown in HFD fed mice does not alter food intake and locomotor activity. S l t fi 17 P d 4 k kd i HFD f d i d ditSupplementary figure 17: Prdm4 knockdown in HFD fed mice decreases energy expenditure.Supplementary figure 18: Effects of Prdm4 knockdown on white and brown adipocyte-selective gene expression in adipose tissues of HFD fed mice. Supplementary figure 19: Effects of Prdm4 knockdown on Ucp1 expression in adipose tissues of HFD fed mice.Supplementary figure 20: Uncropped versions of immunoblots in the figure 1b.Supplementary figure 21: Uncropped versions of immunoblots in the supplementary figures 4b, 7c, and 10d.Supplementary figure 22: Uncropped versions of immunoblots in the supplementary figure 11b and 18b.
Supplementary Notes: Chemical Identity of Butein.Supplementary data set 1: Lists of butein selective genesSupplementary data set 1: Lists of butein selective genes
Nature Chemical Biology: doi:10.1038/nchembio.2081
Number Compound Name Fold change (UCP-1 expression)
1 DMSO 12 BAIBA(3-Aminoisobutyric acid) 0.629923 Resveratrol 0.3965374 r Oryzanol 1 8609224 r-Oryzanol 1.8609225 Quercetin 0.5764026 Butein 7.7882457 Tannic acid 0.5122328 Caffeine 0.7008949 Apigenin 1.03027410 Cyanidin-3-glucoside 0.614538y g11 Peonidin 3-glucoside 1.363312 Fisetin 1.62088813 Ferulic acid 0.57342614 Coumaric acid 0.71662515 Conjugate linoleic acid 1.48918216 Dgat inhibitor 0.62309917 Sesamol 1.74727518 Rutin 1.19985419 Licochalcone A 0.74157920 Glucosamine 0.6241121 Sulfuretin 0.74745922 Glycyrrhizic acid 1.10699423 i b i id 0 62750623 γ-aminobutyric acid 0.62750624 Astaxanthin 0.78045425 β-Carotene 0.70358626 Epigallocatechin gallate 1.88294227 Chitosan 0.93890328 Dimethylfumarate 1.02260529 Mono methyl fumarate 0 8442429 Mono-methyl fumarate 0.8442430 Bisphenol A 0.47781331 Daidzein 1.2023132 Ascorbic acid 0.54204733 Curcumin 0.92893334 WP 1066 2.01060335 Genestein 1.10929736 Luteolin 0.43504637 Cordycephine 0.8775738 Kaempferol 1.14279139 Thymoquinone 1.55605440 Myricetin 1.122241 β-Estradiol 0.751744
Supplemental Table 1. Ucp1 induction by bioactive compounds. C3H10T1/2 adipocytes were treated with bioactive compounds (20 μM) for 6 hours and Ucp1 expression was measured by real time PCR. Fold increase relative to DMSO treatment was shown. r-Oryzanol is a mixture of steryl and other triterpenyl esters of ferulic acids and Conjugated linoleic acids (CLA) is a family of at least 28 isomers of linoleic acid.
42 Tert-butylhydroquinone 0.881284
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Supplementary Figure 1. Identification of butein as an Ucp1 inducer. (a) C3H10T1/2 adipocytes were treated with various polyphenols isolated from herbal products, including the known anti-adipogenic compounds resveratrol, butein, and sulfuretin. Ucp1 mRNA expression was measured by real time PCR. Data represent means ± s.d. (n=3). (b) C3H10T1/2 adipocytes were treated with butein, resveratrol or sulfuretinfor 6 hours and the gene expression profiles were analyzed by microarray. Diagram showing the number of genes (> 1.6 fold) regulated by g p p y y y g g g ( ) g ybutein, sulfuretin (Sul), and resveratrol (Res). (c) Butein but not resveratrol and sulfuretin induces Prdm4 mRNA expression. Data represent means ± s.d. (n=3). Statistical significance was determined relative to a control by the Student’s t-test (* P<0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Supplementary Figure 3. Butein induces expression of thermogenic genes in adipocytes. (a) Mitochondrial staining by mitochondria specific cytopainter (ab112145, Abcam) in C3H10T1/2 adipocytes (left) and quantified by NIH Image J software (right). Data represent means ± s.d.(n=3). (scale bar = 100 μm) (b) C3H10T1/2 adipocytes were treated with butein and stained with bodipy (green). Quantification of changes in ( ) ( μ ) ( ) p y py (g ) Q glipid droplet (LD) size (right). Data represent means ± s.d. (n=3). (scale bar = 50 μm) (c) Primary adipocytes were treated with butein for 24 hours and expression levels of general adipocyte markers (Pparγ and aP2), white adipocyte-selective markers (Nnmt and Retn), and thermogenic-selective markers (Ucp1, Cidea, Ppargc1α, and Dio2) were measured by realtime PCR. Data represent means ± s.d. (n=3). Statistical significance was determined relative to a control by Student’s t-test (* P<0.05; ** P<0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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confirmed by real time PCR. Data represent means ± s.d. (n=3). Statistically significant differences were determined by Student’s t-test (* P<0.05; ** P<0.005; *** P<0.0005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Supplementary Figure 6 Effects on Ucp1 expression by butein selective genes Two independent siRNA against butein selective genes andSupplementary Figure 6. Effects on Ucp1 expression by butein-selective genes. Two independent siRNA against butein selective genes and the top three genes most induced by butein (Hmox-1, Maff, and Atf3) were transfected into C3H10T1/2 adipocytes and Ucp1 expression was measured by real time PCR. Data represent means ± s.d. (n=3). Statistically significant differences were determined by Student’s t-test (* P<0.05; ** P<0.005; *** P<0.0005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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DMSO Butein Isoproterenol DMSO Butein Isoproterenol
pp y g y pPrdm family members (Prdm2, Prdm3, and Prdm16). Isoproterenol does not regulate all Prdm family members, including Prdm4. Data represent means ± s.d. (n=3). Statistically significant differences were determined by Student’s t-test (** P < 0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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( ) y p p p y ( p γ ) (Retn). Data represent means ± s.d. (n=3). (c) Transient transfection of two independent Prdm4-targeting siRNAs (si#1 and si#2) in C3H10T1/2 cells increases lipid accumulation during adipocyte differentiation. Data are representative of two independent experiments. (d) Knockdown of Prdm4 in C3H10T1/2 cells increases the expression of pan- and WAT-selective adipocyte markers. Data represent means ± s.d. (n=3). Statistically significant differences were determined by Student’s t-test (* P<0.05; ** P<0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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Nature Chemical Biology: doi:10.1038/nchembio.2081
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pp y g p g p p y g pPrdm4 decreases pan- and white-selective genes in 3T3-L1 adipocytes. Data represent means ± s.d. (n=3). Statistically significant differences were determined by Student’s t-test (* P<0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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it h d i l t i i b t t t ith b t i i i d i P d 4 iRNA t f t d C3H10T1/2 di t D t t ± dmitochondrial staining by treatment with butein is compromised in Prdm4 siRNA-transfected C3H10T1/2 adipocytes. Data represent means ± s.d.(n=3). (c) Prdm4-overexpressing cells abrogate the effects of butein in increased mitochondrial staining of C3H10T1/2 cells. Data represent means ± s.d. (n=3). Statistically significant differences in the control and butein treatment were determined by Student’s t-test (* P < 0.05; **P<0.005; *** P<0.0005; NS, not significant).
Nature Chemical Biology: doi:10.1038/nchembio.2081
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ND HFD (n=7) (n=7)
0 0WT db/db
(n=4) (n=4)
0
R
iWATeWAT eWAT2.0
1.5
Prdm4**es
sion
1.0
0.5Rel
ativ
e ex
pre
(Fol
d)
ND HFD (n=7) (n=7)
0
R
Supplementary Figure 14. Prdm4 expression is reduced in adipose tissues of obese and diabetic mice. (a) Prdm4 expression in lean and obese fat tissues. Prdm4 expression is significantly lower in epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT), and brown adipose tissues (BAT) of the HFD-fed obese mice compared to the lean control mice (n=7 per group). Dots (open and closed) and
BAT
bars in the scatter plots represent individual mice and the average, respectively. (b) The expression levels of Prdm4 in epididymal fats of diabetic mice (db/db) are compared to non-diabetic control mice (n=4 per group). Dots (open and closed) and bars in the scatter plots represent individual mice and the average, respectively. Statistically significant differences in the control and butein treatment were determined by Student’s t-test (* P < 0.05; ** P<0.005; *** P<0.0005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
Ctrl ASO Prdm4 ASO
Ctrl ASO Prdm4 ASO
b180
160l)
a
2.5
C l P d 4
Ctrl Prdm4Ctrl Prdm4
* P=0.1140
120luco
se (m
g/d
2.0
1.5
1 0Wei
ght (
g)
* Ctrl Prdm4
0 30 60 90 120 (min)
100
80
Gl1.0
0.5
0
W
0 30 60 90 120 (min)0Fat Liver
S l t Fi 15 Eff t f P d 4 k kd ti i ht i d i li iti it i HFD f d i ( b) C57BL/6Supplementary Figure 15. Effects of Prdm4 knockdown on tissue weight gains and insulin sensitivity in HFD fed mice. (a-b) C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. (a) Liver and fat weights of C57BL/6 mice treated with control ASO or Prdm4 ASO. Data represent means ± s.d. (n=5). (b) Insulin tolerance test in control and Prdm4 ASO-injected groups. Data represent means ± s.d. (n=5). Statistically significant differences in the control ASO and Prdm4 ASO-injected groups were determined by Student’s t-test (* P < 0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
ae
4
3NS
b6000
nts
se)
NSly
food
inta
ke /
mou
se)
3
2
4000
2000Act
ivity
cou
n(d
ay/m
ous
Dai (g 1
0
2000
0Ctrl ASO Prdm4 ASO
( 5) ( 5)Ctrl ASO Prdm4 ASO
( 5) ( 5)(n=5) (n=5) (n=5) (n=5)
Supplementary Figure 16. Prdm4 knockdown in HFD fed mice does not alter food intake and locomotor activity. C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. (a) Food intake of HFD-fed control ASO and Prdm4 ASO-treated mice. Data represent means ± s.d. (n=5). (b) Physical activity of HFD-fed control ASO and Prdm4 ASO-treatedASO and Prdm4 ASO treated mice. Data represent means ± s.d. (n 5). (b) Physical activity of HFD fed control ASO and Prdm4 ASO treated mice. Data represent means ± s.d. (n=5). Statistically significant differences in the control ASO and Prdm4 ASO-injected groups were determined by Student’s t-test (NS, not significant).
Nature Chemical Biology: doi:10.1038/nchembio.2081
b
48 Ctrl ASOPrdm4 ASO
min
/kg)
55
50
Ctrl ASO Prdm4 ASO
a
min
/kg)
44
40
umpt
ion(
ml/m
45
40*
umpt
ion(
ml/m
36
32Dark light
VO2
Con
su 35
30
25
Dark light
*
VO2
Con
s
Dark light25
40
kg)
Ctrl ASO Prdm4 ASO 34
Ctrl ASOPrdm4 ASO n/
kg)
c d
35
30
ptio
n(m
l/min
/k
30
**mpt
ion(
ml/m
in
25
20
CO
2C
onsu
mp
Dark light
26
22
**
VCO
2C
onsu
m
15
VC
Dark light 22Dark light
VSupplementary Figure 17. Prdm4 knockdown in HFD fed mice decreases energy expenditure. (a-d) Mice on a high fat diet were treated with Prdm4 ASO or control ASO for 2.5 weeks and energy expenditure was measured. Data represent means ± s.d. (n=5). (a) O2 consumption wasPrdm4 ASO or control ASO for 2.5 weeks and energy expenditure was measured. Data represent means ± s.d. (n 5). (a) O2 consumption was recorded during a 24 hour period. (b) averages of O2 consumption. (c) CO2 production was recorded during a 24 hour period. (d) averages of CO2production. Statistically significant differences in the control and Prdm4 ASO-treated mice were determined by Student’s t-test (* P < 0.05; **P<0.005).
Nature Chemical Biology: doi:10.1038/nchembio.2081
a
Prdm162 0
Cidea2 0
Nnmt2 0
bBrown adipocyte White adipocyte
)*
2.0
1.5
*2.0
1.5
P=0.142.0
1.5
)
essi
on (F
old) 1.0
0.5
1.0
0.5
1.0
0.5
essi
on (F
old)
Rel
ativ
e ex
pre
0 0Ppargc1α
P=0.062.0
Cox7a1
*2.0
0Retn
*2.0
Rel
ativ
e ex
pre
R
1.5
1.0
1.5
1.0
1.5
1.0
R
0.5
0
0.5
0
0.5
0Ctrl ASO Prdm4 ASOCtrl ASO Prdm4 ASOCtrl ASO Prdm4 ASO Ctrl ASO Prdm4 ASO
(n=6) (n=6) Ctrl ASO Prdm4 ASO
(n=6) (n=6) Ctrl ASO Prdm4 ASO
(n=6) (n=6)
Supplementary Figure 18. Effects of Prdm4 knockdown on white and brown adipocyte selective gene expression in adipose tissues of HFD fed mice. (a-b) C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. Expression of brown (a) and white adipocyte-selective genes (n=6 per group) (b) in epididymal adipose tissues of control and Prdm4 ASO-treated mice (n=6 per group). Dots (open and closed) and bars in the scatter plots represent individual mice and the average, respectively. Statistically significant differences in the control ASO and Prdm4 ASO-injected groups were determined by Student’s t-test (* P < 0.05).
Nature Chemical Biology: doi:10.1038/nchembio.2081
P d 4 ASOa b
Prdm4 ASOCtrl ASO BAT
Ucp1
Prdm42.0
1.5
2.0
1.5
Prdm4 Ucp1
ion
(Fol
d)
** *
Ucp1
β-Actin1.0
0.5
1.0
0.5ativ
e ex
pres
si
Longer exposure
Ucp1
0.5
0
0.5
0
Rel
a
Ctrl ASO Prdm4 ASO(n=5) (n=5)
Ctrl ASO Prdm4 ASO(n=5) (n=5)
S l t Fi 19 Eff t f P d 4 k kd U 1 i i di ti f HFD f d i ( b) C57BL/6 iSupplementary Figure 19. Effects of Prdm4 knockdown on Ucp1 expression in adipose tissues of HFD fed mice. (a-b) C57BL/6 mice on a HFD were treated with Prdm4 ASO or a control ASO twice per week (25mg per kg per dose) for 6 weeks. (a) Expression of Prdm4 and Ucp1in epididymal adipose tissues (eWAT) of control and Prdm4 ASO-injected mice (n=5 per group). Dots (open and closed) and bars in the scatter plots represent expression levels of individual mice and the average, respectively. (b) Expression of Prdm4 and Ucp1 protein in eWAT of control ASO and Prdm4 ASO-injected mice (n=5 per group) was determined by western blotting. Brown adipose tissues (BAT) were used as controls. Uncropped images of blots are shown in Supplementary Fig. 22.
Nature Chemical Biology: doi:10.1038/nchembio.2081
Original blots
- + - +12h 24h
Butein - + - +12h 24h
Fig. 1b
- + - +12h 24h
(kD )Butein Butein
170100
7055403525
(kDa)170100
70554035
(kDa) 170100
7055403525
(kDa)
WB: Ucp1 WB: ActinWB: Prdm4
25
1025
1010
Supplementary Figure 20 Uncropped versions of immunoblots in the Fig 1bSupplementary Figure 20. Uncropped versions of immunoblots in the Fig 1b.
Nature Chemical Biology: doi:10.1038/nchembio.2081
Original blots
Supplementary Fig.4b
DMSO 10 20 (μM)
ButeinDMSO 10 20 (μM)
Butein
DMSO 10 20 (μM) Butein
(Kd )
Supplementary Fig 10.d
scr si#1 si#2
Prdm4
i#1 i#2
PRDM4(kD ) scr si#1 si#2
Prdm4(μ ) (μ )
170100
7055403525
(Kda)170100
7055403525
(Kda)170100
7055403525
(Kda)scr si#1 si#2scr si#1 si#2
170100
7055403525
(kDa)170100
7055403525
(kDa)170100
705540
35
(kDa) scr si#1 si#2
WB: Prdm4 WB: Ucp1 WB: Actin
25
10 1025
10
WB: ActinWB: Prdm4
25
10
25
1025
WB: Ucp1
Supplementary Fig.7c
(Kd )ctrl Butein (15mg/kg) ctrl Butein (15mg/kg) ctrl Butein (15mg/kg)
170100
70
5540
(Kda)170100
70
55
40
(Kda)170100
70
5540
35
(Kda)
3525
35
25
35
25
WB: Prdm4 WB: Ucp1 WB: Actin
Supplementary Figure 21 Uncropped versions of immunoblots in the supplementary Figure 4b, 7c, and 10d
Nature Chemical Biology: doi:10.1038/nchembio.2081
Original blots
Ctrl ASO Prdm4 ASO BAT
Supplementary Fig 19. b Supplementary Fig.11b
scr si#1 si#2Prdm4
170
(Kda)
170
100705540
(kDa)
WB: Prdm4
170100
7055403525
103525
(kDa)
170
100
Longer exposure: Ucp1WB: Prdm4
scr si#1 si#2Prdm4
10
705540
3525
scr si#1 si#2170100
7055403525
(Kda)
(kDa)
WB A ti
WB: Ucp1170
1007055
WB: Ucp1
Prdm4
10
WB: Actin403525
scr si#1 si#2Prdm4
170100
705540
(Kda)
Supplementary Figure 22. Uncropped versions of immunoblots in the supplementary figure 11b and 18b.WB: Actin
3525
10
Nature Chemical Biology: doi:10.1038/nchembio.2081