supplementary figure 1 a b - nature · supplementary figure 1. ... supplementary figure 2 ... il-1b...
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![Page 1: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/1.jpg)
Supplementary Figure 1
0.0
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b �
Trans 1 Trans 2 Trans 3 0.0
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0.4 iWAT adipocytes gWAT adipocytes
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Trans 1 Trans 2 Trans 3 0.0
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0.6 Trip-br2 transcripts iWAT gWAT
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4 F4/80
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SVF Adipose tissue
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gWAT
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SVF Adipose tissue 0
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HFD *
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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.
![Page 2: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/2.jpg)
Ctrl Tuni-6h Tuni-12h Tuni-18h
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Bip
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Chop
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Chop Atf4 Xbp1s Creb3 0.0
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## * * *
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Chop Atf4 Xbp1s Creb3 0.0
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iWAT gWAT Liver Heart 0
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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
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* *
** Chop
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Human adipocytes-PPARγ2
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Visceral SQ Rel
ativ
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.U.)
f e e
gWAT iWAT 0.0 0.5 1.0 1.5 2.0 400 500 600 700 800
Pparγ
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Human adipocytes-AP2 Undifferentiated Differentiated
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Visceral SQ Rel
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/TBP
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.)
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).
![Page 3: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/3.jpg)
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Il-1b Il-6 Ifnγ Tnfa Mcp1 F4/80 Crp Saa Sap
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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.
![Page 4: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/4.jpg)
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GFP
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CH
OP
-V5 �
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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.
![Page 5: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/5.jpg)
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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.
![Page 6: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/6.jpg)
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
![Page 7: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/7.jpg)
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
![Page 8: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/8.jpg)
Supplementary Table 1: Compounds concentration for Figure 2a Compound Concentration TNFa 100 ng/ml MCP1 100 ng/ml IFNg 10 µg/ml IL6 2 µg/ml IL10 100 ng/ml BSA 0.5 M Palmitate 0.2 M Insulin 100 nM IL1β 25 ng/ml IL15 100 ng/ml CRP 200 ng/ml PAI-1 500 ng/ml Leptin 100 ng/ml Adiponectin 20 µg/ml
![Page 9: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/9.jpg)
Supplementary Table 2. Primer sequences used in this study Gene Primer sequence
Forward: 5'- GCT CTC ACT CGT CGG GAT CC -3' mTrip-br2-tran1
Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -3' Forward: 5'- GAA GGC TTT GTG CAC GTG AGG -3' mTrip-br2-tran2
Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -3' Forward: 5'- GCT CCC TGC TAC CGT CGG C -3' mTrip-br2-tran3
Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -3' Forward: 5'- ATA TAT GTT GGG TAA AGG AGG AA -3' mTrip-br2-ORF
Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -3' Forward: 5'- TGG AGA GTG TGG ATC CCA AGC AA T-3' mIl-1b
Reverse: 5'- TGT CCT GAC CAC TGT TGT TTC CCA -3' Forward: 5'- ATC CAG TTG CCT TCT TGG GAC TGA -3' mIl-6
Reverse: 5'- TAA GCC TCC GAC TTG TGA AGT GGT -3' Forward: 5'- TCT GGA GGA ACT GGC AAA AG -3' mIfnγ
Reverse: 5'- TTC AAG ACT TCA AAG AGT CTG AGG -3' Forward: 5'- CCA CTC ACC TGC TGC TAC TCA T -3' mMcp-1
Reverse: 5'- TGG TGA TCC TCT TGT AGC TCT CC -3' Forward: 5'- GGG ACG AAA CTG GAG ATG TTA T -3' mPAI-1
Reverse: 5'- GAG GAG TTG CCT TCT CTT TCT C -3' Forward: 5'- GCC TCT TCT CAT TCC TGC TTG T -3' mTnfa
Reverse: 5'- GGC CAT TTG GGA ACT TCT CAT -3' Forward: 5'- GGC GGG CAC TGA ACT ATA AA -3' mCrp
Reverse: 5'- CGA GAC AAG GGA GAG AAT GTA ATC -3' Forward: 5'- CTA GGA ACA CTG AAG ATG CTC TC -3' mSaa1/2
Reverse: 5'- TCT CCT CCT CAA GCA GTT ACT A -3' Forward: 5'- TGT CTT CAC CAG CCT TCT TTC -3' mSap
Reverse: 5'- CTA GAT GTG GGA TCA GCT TCA C -3' Forward: 5'- TTT CCT CGC CTG CTT CTT C -3' mF4/80
Reverse: 5'- CCC CGT CTC TGT ATT CAA C -3' Forward: 5'- TTC AGC CAA TTA TCA GCA AAC TCT -3' mBiP
Reverse: 5'- TTT TCT GAT GTA TCC TCT TCA CCA GT -3' Forward: 5'- CCA CCA CAC CTG AAA GCA GAA -3' mChop
Reverse: 5'- AGG TGA AAG GCA GGG ACT CA -3' Forward: 5'- CTG AGT CCG AAT CAG GTG CAG -3' mXBP1s
Reverse: 5'- GTC CAT GGG AAG ATG TTC TGG -3' Forward: 5'- TTC CGG GAC AGA TTG GAT GTT GGA -3' mAtf4
Reverse: 5'- ATG GCC AAT TGG GTT CAC TGT CTG -3' Forward: 5'- CTG GGC TCG GTA GTT TGT ATC -3' mAtf6
Reverse: 5'- AGA CCT GAA TGG CTG CTT AC -3' Forward: 5'- CCA CTC TGG GAT CTT TCT TCA G -3' mCreb3
Reverse: 5'- AAA GAA GGG CTG TGG TTA GG -3' mGata1 Forward: 5'- ACT AAC TGT CAA ACG ACC ACT AC-3'
![Page 10: 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](https://reader030.vdocument.in/reader030/viewer/2022040622/5d2150eb88c993ea218ddd09/html5/thumbnails/10.jpg)
Reverse: 5'- TCA CCT GAT GGA GCT TGA AAT AG -3' Forward: 5'- GGA GAA AGG AGT AGG CAA GAA G -3' mGata2
Reverse: 5'- CCC AAG AAC ACA AAT AGC ACA C -3' Forward: 5'- AGC TGC CAG ATA GCA TGA AG -3' mGata3
Reverse: 5'- TAG GGC GGA TAG GTG GTA AT -3' Forward: 5'- ACC CTT CAC CAA TGA CTC CTA TG -3' mTbp
Reverse: 5'- ATG ATG ACT GCA GCA AAT CGC -3' mPparγ Forward: 5'- TCG CTG ATG CAC TGC CTA TG Reverse: 5'- GAG AGG TCC ACA GAG CTG ATT
Forward: 5'- TGA TGC CTT ATG GCA CTG GAC TCA-3' human TBP
Reverse: 5'- CTG CTG CCT TTG TTG CTC TTC CAA-3' Forward: 5'- GAC TCT CTG CCT GGG AAT TT -3' human TRIP-BR2-
ORF Reverse: 5'- GGA CGT ATC AAT GTC AGC AAA C -3' Forward: 5'- CTC GGA GGG TTT CTT GTT TCT -3' human GATA3
Reverse: 5'- CAG GAA TAG GGA CAA GAC AGA TG-3' Forward: 5'- AGA GTG AGA CTC CAC CTC AA -3' human BIP
Reverse: 5'- CAG CCA GTT GCC CAT CTA TAA -3' human PPARγ Forward: 5'- AGC CTC ATG AAG AGC CTT CCA Reverse: 5'- TCC GGA AGA AAC CCT TGC A human AP2 Forward: 5'- CGT CAC TTC CAC GAG AGT TTA T Reverse: 5'- TCC CAC AGA ATG TTG TAG AGT TC