Supplementary Figure 1 0.0 0.5 1.0 1.5 Trip-br2 Ctrl LPS Relative expression/TBP (A.U.) b Trans 1 Trans 2 Trans 3 0.0 0.1 0.2 0.3 0.4 iWAT adipocytes gWAT adipocytes Trip-br2 transcripts Normalized expression/TBP (A.U.) Trans 1 Trans 2 Trans 3 0.0 0.2 0.4 0.6 Trip-br2 transcripts iWAT gWAT Normalized expression/TBP (A.U.) a 0 1 2 3 4 F4/80 Relative expression/TBP (A.U.) SVF Adipose tissue iWAT SVF Adipose tissue gWAT c SVF Adipose tissue 0 10 20 30 40 F4/80 CD HFD * Relative expression/TBP (A.U.) gWAT *** Supplementary Figure 1. TRIP-Br2 expression in WAT and adipocytes treated with LPS (a) qPCR analysis of TRIP-Br2 transcripts gene expression in gonadal (gWAT) or inguinal (iWAT) adipose tissues or isolated mature adipocyted from chow diet-fed mice (n=6 per group replicated twice). (b) qPCR analysis of TRIP-Br2 gene expression in 3T3-L1 differentiated adipocytes treated with vehicle or LPS for 24 h (n=4 per group). (c) qPCR analysis of F4/80 gene expression in gWAT or iWAT stromal-vascular-fraction (SVF) or adipose tissues from CD or HFD-fed mice (n=5 per group replicated twice). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, *,p<0.05; **,p<0.01; ***,p<0.001.
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Supplementary Figure 1 a b - Nature · Supplementary Figure 1. ... Supplementary Figure 2 ... Il-1b Il-6 Ifnγ Tnfa Mcp1 F4/80 Crp Saa Sap iWAT WT-Ctrl WT-Tuni KO-Ctrl
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Supplementary Figure 1
0.0
0.5
1.0
1.5 Trip-br2
Ctrl LPS
Rel
ativ
e ex
pres
sion
/TBP
(A.U
.)
b �
Trans 1 Trans 2 Trans 3 0.0
0.1
0.2
0.3
0.4 iWAT adipocytes gWAT adipocytes
Trip-br2 transcripts
Nor
mal
ized
exp
ress
ion/
TBP
(A.U
.)
Trans 1 Trans 2 Trans 3 0.0
0.2
0.4
0.6 Trip-br2 transcripts iWAT gWAT
Nor
mal
ized
exp
ress
ion/
TBP
(A.U
.) a �
0
1
2
3
4 F4/80
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
SVF Adipose tissue
iWAT
SVF Adipose tissue
gWAT
c
SVF Adipose tissue 0
10
20
30
40 F4/80 CD
HFD *
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
gWAT
***
Supplementary Figure 1. TRIP-Br2 expression in WAT and adipocytes treated with LPS (a) qPCR analysis of TRIP-Br2 transcripts gene expression in gonadal (gWAT) or inguinal (iWAT) adipose tissues or isolated mature adipocyted from chow diet-fed mice (n=6 per group replicated twice). (b) qPCR analysis of TRIP-Br2 gene expression in 3T3-L1 differentiated adipocytes treated with vehicle or LPS for 24 h (n=4 per group). (c) qPCR analysis of F4/80 gene expression in gWAT or iWAT stromal-vascular-fraction (SVF) or adipose tissues from CD or HFD-fed mice (n=5 per group replicated twice). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, *,p<0.05; **,p<0.01; ***,p<0.001.
Ctrl Tuni-6h Tuni-12h Tuni-18h
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ativ
e ex
pres
sion
/TB
P (A
.U.) Atf4
iWAT gWAT 0
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4
6
8
10
*** ***
***
***
***
Bip
iWAT gWAT 0
1
2
3
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5
*
* **
Chop
iWAT gWAT 0
2
4
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* *
***
**
*
CD HFD
HFD+TUDCA
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
Chop Atf4 Xbp1s Creb3 0.0
0.5
1.0
1.5
2.0
2.5 ***
## * * *
a
b
c d
#
CD-4 wk
HFD-4 wk CD-8 wk HFD-8 wk
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ativ
e ex
pres
sion
/TB
P (A
.U.)
Chop Atf4 Xbp1s Creb3 0.0
0.5
1.0
1.5
2.0
2.5
* * * *
* ** *
Atf4 Ctrl Tuni
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
iWAT gWAT Liver Heart 0
1
2
3
*** ***
**
Bip
iWAT gWAT Liver Heart 0 1 2 3 4 5 8
10 12 14
*
*
***
iWAT gWAT Liver Heart 0 1 2 3 4 5
20 25 30 35 40
* *
** Chop
0
1
2
3 **
Human adipocytes-PPARγ2
**
Visceral SQ Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
f e e
gWAT iWAT 0.0 0.5 1.0 1.5 2.0 400 500 600 700 800
Pparγ
*** ***
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
0.0
0.5
1.0
1.5
2.0 100 200 300 400
**
Human adipocytes-AP2 Undifferentiated Differentiated
**
Visceral SQ Rel
ativ
e ex
pres
sion
/TBP
(A.U
.)
Supplementary Figure 2
Supplementary Figure 2. ER stress markers induced by chemical or high-fat diet qPCR analysis of ER stress markers gene expression in (a) tissues harvested from mice intraperitoneally (IP) injected with vehicle or tunicamycin (2.5 mg/kg) for 18 h (n=5 per group replicated thrice); (b) iWAT or gWAT from mice IP injected with vehicle or tunicamycin (2.5 mg/kg) for 6, 12 or 18 h (n=5 per group replicated twice); (c) gWAT from mice after 12 wk of CD, HFD or HFD with TUDCA (250 mg/kg at 8am and 8pm, i.p., total 500 mg/kg for 15 days) (n=5 per group); (d) gWAT from mice after 4 or 8 wk of CD or HFD (n=5 per group). qPCR analysis of PPARγ or AP2 gene expression in (e) undifferentiated or differentiated gWAT or iWAT preadipocytes or adipocytes (n=3 per group replicated twice); (f) undifferentiated or differentiated human visceral or subcutaneous preadipocytes or adipocytes (n=3 per group replicated twice). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, *,p<0.05; **,p<0.01; ***,p<0.001 (vs CD); #, p<0.01; ##,p<0.01 (vs HFD).
0
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6
** *** ***
**
*
**
Il-1b Il-6 Ifnγ Tnfa Mcp1 F4/80 Crp Saa Sap
iWAT WT-Ctrl WT-Tuni KO-Ctrl KO-Tuni
a
b
Rel
ativ
e ex
pres
sion
/TB
P (A
.U)
0
1
2
*** *** * *** **
** ** **
Sap Il-1b Il-6 Ifnγ Tnfa Mcp1 Crp Saa
WT-Ctrl WT-Tuni
KO-Ctrl KO-Tuni
iWAT adipocytes
Rel
ativ
e ex
pres
sion
/TB
P (A
.U)
12 wk HFD gWAT iWAT
Il-1b Il-6 Ifnγ Tnfa Crp Saa Sap 0.0
0.5
1.0
1.5
*** *** *** * *
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ativ
e ex
pres
sion
/TB
P (A
.U.)
Pai-1
c
*** *** ***
*** *** **
***
***
12 wk HFD iWAT
Il-1b Il-6 Ifnγ Pai-1 Tnfa Crp Saa Sap 0.0
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1.0
1.5 2.0 3.0 4.0 WT
KO
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ativ
e ex
pres
sion
/TB
P (A
.U.)
d
Supplementary Figure 3
Supplementary Figure 3. Expression of inflammatory and acute phase response markers in WAT qPCR analysis of inflammation and acute phase response markers gene expression in (a) iWAT from TRIP-Br2 WT or KO mice after 18 h of vehicle or tunicamycin (2.5 mg/kg, i.p.) treatment (n=5 per group replicated twice); (b) iWAT adipocytes differentiated from primary WT or KO SVF treated with or without tunicamycin (1 µg/ml) for 24 h (n=3 per group); (c) gWAT or iWAT from WT mice fed with 12 wk of HFD (n=6 per group replicated twice). (d) iWAT from WT or KO mice fed with 12 wk of HFD (n=6 per group replicated twice). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, *,p<0.05; **,p<0.01; ***,p<0.001.
0
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15 Creb3
***
GFP GFP-Creb3-V5
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pres
sion
/TB
P (A
.U.)
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Chop
*** GFP Chop-V5
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ativ
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pres
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/TB
P (A
.U.)
GFP
CR
EB
3-G
FP-V
5
GFP
CH
OP
-V5 �
140–
100–
70–
50–
40–
35–
25–
α-V5
Supplementary Figure 4
Supplementary Figure 4. Expression levels of CREB3 and CHOP qPCR analysis of CREB3 and CHOP in adipocytes overexpressing GFP control, CREB-GFP-V5 or CHOP-V5 (n=3 per group). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, ***,p<0.001; Western blot analysis for CREB or CHOP levels using anti-V5 antibody in adipocytes overexpressing GFP control, CREB-GFP-V5 or CHOP-V5.
0
1
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4
Trip-br2
Ctrl Tuni
pBabe- Neo
pBabe- mGATA3
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ativ
e ex
pres
sion
/TB
P (A
.U.)
b
* �
0
10
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Nor
mal
ized
fold
enr
ichm
ent
Distal Proximal TRIP-Br2 promoter primers
*
*
d Ctrl-IgG Ctrl-Histone
0
20
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60 *
Gata3 pBABE-Neo pBABE-GATA3
Rel
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e ex
pres
sion
/TBP
(A.U
.)
a
0.0
0.5
1.0
1.5 Gata3fl/fl
Gata3
**
Rel
ativ
e ex
pres
sion
/TB
P (A
.U.)
Gata3KO
c
iWAT adipocytes
Supplementary Figure 5
Supplementary Figure 5. Role of GATA3 on TRIP-Br2 expression (a) qPCR analysis of GATA3 in gWAT adipocytes infected with control or GATA3 retrovirus (n=3 per group); (b) qPCR analysis of TRIP-Br2 in iWAT adipocytes infected with control or GATA3 retrovirus with or without tunicamycin treatment (n=3 per group). (c) qPCR analysis of GATA3 gene expression in gWAT adipocytes differentiated from GATA3fl/fl-CreERT2 primary SVF with or without tamoxifen (0.5 mM) treatment to induce GATA3 KO (n=5 per group). (d) qPCR analysis of proximal or distal genomic region of TRIP-Br2 transcript 3 promoter IgG (negative control) or anti-histone (positive control) immunoprecipitation (n=3 per group replicated twice). All qPCR data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test, *,p<0.05; **,p<0.01; ***,p<0.001.
BiP
TRIP-Br2
6h 24h 48h
C T C T C T
3T3-L1 adipocytes
6h 24h 48h
C T C T C T
3T3-L1adipocytes
Supplementary Figure 6
β-actin
6h 24h 48h
C T C T C T
3T3-L1adipocytes
Supplementary Figure 6. Uncropped images of blots shown in Figure 2f
Ctrl 6h 12h 18h
Tuni
Ctrl 6h 12h 18h
Tuni
gWAT iWAT
Ctrl 6h 12h 18h
Tuni
Ctrl 6h 12h 18h
Tuni
gWAT iWAT
BiP
TRIP-Br2
Ctrl 6 12 18
Tuni
Ctrl 6 12 18
Tuni gWAT iWAT
β-actin
Supplementary Figure 7
Supplementary Figure 7. Uncropped images of blots shown in Figure 3e