Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans Trans F/8 SVF Adipose tissue SVF Adipose tissue adipocytes F/8 CD HFD SVF Adipose tissue Supplementary Figure. TRIP-Br expression in WAT and adipocytes treated with LPS (a) qpcr analysis of TRIP-Br transcripts gene expression in gonadal () or inguinal () adipose tissues or isolated mature adipocyted from chow diet-fed mice (n=6 per group replicated twice). (b) qpcr analysis of TRIP-Br gene expression in T-L differentiated adipocytes treated with vehicle or LPS for h (n= per group). (c) qpcr analysis of F/8 gene expression in or 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<.5;,p<.;,p<..
Supplementary Figure a b c 8 6...5. Ctrl.5 Ctrl -6h -h -8h Atf Atf CD HFD HFD+TUDCA ## Liver Heart 8 5 Chop Atf Xbps Creb 5 # d.5...5. Bip Bip Liver Heart CD- wk HFD- wk 5 5 5 8 6 CD-8 wk HFD-8 wk Chop Atf Xbps Creb Liver Heart Chop Chop e 8 7 6 5...5. Pparγ f Human adipocytes-pparγ Visceral SQ...5. Human adipocytes-ap Visceral SQ Undifferentiated Differentiated Supplementary Figure. 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 (.5 mg/kg) for 8 h (n=5 per group replicated thrice); (b) or from mice IP injected with vehicle or tunicamycin (.5 mg/kg) for 6, or 8 h (n=5 per group replicated twice); (c) from mice after wk of CD, HFD or HFD with TUDCA (5 mg/kg at 8am and 8pm, i.p., total 5 mg/kg for 5 days) (n=5 per group); (d) from mice after or 8 wk of CD or HFD (n=5 per group). qpcr analysis of PPARγ or AP gene expression in (e) undifferentiated or differentiated or preadipocytes or adipocytes (n= per group replicated twice); (f) undifferentiated or differentiated human visceral or subcutaneous preadipocytes or adipocytes (n= per group replicated twice). All qpcr data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test,,p<.5;,p<.;,p<. (vs CD); #, p<.; ##,p<. (vs HFD).
Supplementary Figure a Relative expression/tbp (A.U) b 6 WT-Ctrl WT- KO-Ctrl KO- Il-b Il-6 Ifnγ Tnfa Mcp F/8 Crp Saa Sap adipocytes Relative expression/tbp (A.U) WT-Ctrl WT- KO-Ctrl KO- Il-b Il-6 Ifnγ Tnfa Mcp Crp Saa Sap c..5. wk HFD Il-b Il-6 Ifnγ Tnfa Crp Saa Sap Pai- d.....5. WT KO wk HFD Il-b Il-6 Ifnγ Tnfa Crp Saa Sap Pai- Supplementary Figure. Expression of inflammatory and acute phase response markers in WAT qpcr analysis of inflammation and acute phase response markers gene expression in (a) from TRIP-Br WT or KO mice after 8 h of vehicle or tunicamycin (.5 mg/kg, i.p.) treatment (n=5 per group replicated twice); (b) adipocytes differentiated from primary WT or KO SVF treated with or without tunicamycin ( µg/ml) for h (n= per group); (c) or from WT mice fed with wk of HFD (n=6 per group replicated twice). (d) from WT or KO mice fed with 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<.5;,p<.;,p<..
Supplementary Figure 5 5 Creb GFP GFP-Creb-V5 8 6 Chop GFP Chop-V5 α-v5 7 5 5 5 GFP CREB-GFP-V5 GFP CHOP-V5 Supplementary Figure. Expression levels of CREB and CHOP qpcr analysis of CREB and CHOP in adipocytes overexpressing GFP control, CREB-GFP-V5 or CHOP-V5 (n= per group). All qpcr data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test,,p<.; Western blot analysis for CREB or CHOP levels using anti-v5 antibody in adipocytes overexpressing GFP control, CREB-GFP-V5 or CHOP-V5.
Supplementary Figure 5 a 6 Gata pbabe-neo pbabe-gata b Trip-br Ctrl pbabe- pbabe- Neo mgata adipocytes c..5. Gata Gatafl/fl GataKO d Normalized fold enrichment 6 5 Ctrl-IgG Ctrl-Histone Distal Proximal TRIP-Br promoter primers Supplementary Figure 5. Role of GATA on TRIP-Br expression (a) qpcr analysis of GATA in adipocytes infected with control or GATA retrovirus (n= per group); (b) qpcr analysis of TRIP-Br in adipocytes infected with control or GATA retrovirus with or without tunicamycin treatment (n= per group). (c) qpcr analysis of GATA gene expression in adipocytes differentiated from GATAfl/fl-CreERT primary SVF with or without tamoxifen (.5 mm) treatment to induce GATA KO (n=5 per group). (d) qpcr analysis of proximal or distal genomic region of TRIP-Br transcript promoter IgG (negative control) or anti-histone (positive control) immunoprecipitation (n= per group replicated twice). All qpcr data are normalized with TBP and presented as mean ± SEM. Two-tailed student t-test,,p<.5;,p<.;,p<..
Supplementary Figure 6 T-L adipocytes 6h h 8h C T C T C T T-L adipocytes TRIP-Br 6h h 8h C T C T C T BiP T-L adipocytes 6h h 8h C T C T C T β-actin Supplementary Figure 6. Uncropped images of blots shown in Figure f
Supplementary Figure 7 Ctrl 6h h 8h Ctrl 6h h 8h TRIP-Br BiP Ctrl 6h h 8h Ctrl 6 8 Ctrl 6 8 β-actin Supplementary Figure 7. Uncropped images of blots shown in Figure e Ctrl 6h h 8h
Supplementary Table : Compounds concentration for Figure a Compound TNFa MCP IFNg IL6 IL BSA Palmitate Insulin ILβ IL5 CRP PAI- Leptin Adiponectin Concentration ng/ml ng/ml µg/ml µg/ml ng/ml.5 M. M nm 5 ng/ml ng/ml ng/ml 5 ng/ml ng/ml µg/ml
Supplementary Table. Primer sequences used in this study Gene mtrip-br-tran mtrip-br-tran mtrip-br-tran mtrip-br-orf mil-b mil-6 mifnγ mmcp- mpai- mtnfa mcrp msaa/ msap mf/8 mbip mchop mxbps matf matf6 mcreb mgata Primer sequence Forward: 5'- GCT CTC ACT CGT CGG GAT CC -' Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -' Forward: 5'- GAA GGC TTT GTG CAC GTG AGG -' Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -' Forward: 5'- GCT CCC TGC TAC CGT CGG C -' Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -' Forward: 5'- ATA TAT GTT GGG TAA AGG AGG AA -' Reverse: 5'- TGG CGC TGT AAG GTG TAA GAC -' Forward: 5'- TGG AGA GTG TGG ATC CCA AGC AA T-' Reverse: 5'- TGT CCT GAC CAC TGT TGT TTC CCA -' Forward: 5'- ATC CAG TTG CCT TCT TGG GAC TGA -' Reverse: 5'- TAA GCC TCC GAC TTG TGA AGT GGT -' Forward: 5'- TCT GGA GGA ACT GGC AAA AG -' Reverse: 5'- TTC AAG ACT TCA AAG AGT CTG AGG -' Forward: 5'- CCA CTC ACC TGC TGC TAC TCA T -' Reverse: 5'- TGG TGA TCC TCT TGT AGC TCT CC -' Forward: 5'- GGG ACG AAA CTG GAG ATG TTA T -' Reverse: 5'- GAG GAG TTG CCT TCT CTT TCT C -' Forward: 5'- GCC TCT TCT CAT TCC TGC TTG T -' Reverse: 5'- GGC CAT TTG GGA ACT TCT CAT -' Forward: 5'- GGC GGG CAC TGA ACT ATA AA -' Reverse: 5'- CGA GAC AAG GGA GAG AAT GTA ATC -' Forward: 5'- CTA GGA ACA CTG AAG ATG CTC TC -' Reverse: 5'- TCT CCT CCT CAA GCA GTT ACT A -' Forward: 5'- TGT CTT CAC CAG CCT TCT TTC -' Reverse: 5'- TA GAT GTG GGA TCA GCT TCA C -' Forward: 5'- TTT CCT CGC CTG CTT CTT C -' Reverse: 5'- CCC CGT CTC TGT ATT CAA C -' Forward: 5'- TTC AGC CAA TTA TCA GCA AAC TCT -' Reverse: 5'- TTT TCT GAT GTA TCC TCT TCA CCA GT -' Forward: 5'- CCA CCA CAC CTG AAA GCA GAA -' Reverse: 5'- AGG TGA AAG GCA GGG ACT CA -' Forward: 5'- CTG AGT CCG AAT CAG GTG CAG -' Reverse: 5'- GTC CAT GGG AAG ATG TTC TGG -' Forward: 5'- TTC CGG GAC AGA TTG GAT GTT GGA -' Reverse: 5'- ATG GCC AAT TGG GTT CAC TGT CTG -' Forward: 5'- CTG GGC TCG GTA GTT TGT ATC -' Reverse: 5'- AGA CCT GAA TGG CTG CTT AC -' Forward: 5'- CCA CTC TGG GAT CTT TCT TCA G -' Reverse: 5'- AAA GAA GGG CTG TGG TTA GG -' Forward: 5'- ACT AAC TGT CAA ACG ACC ACT AC-'
mgata mgata mtbp mpparγ human TBP human TRIP-BR- ORF human GATA human BIP human PPARγ human AP Reverse: 5'- TCA CCT GAT GGA GCT TGA AAT AG -' Forward: 5'- GGA GAA AGG AGT AGG CAA GAA G -' Reverse: 5'- CCC AAG AAC ACA AAT AGC ACA C -' Forward: 5'- AGC TGC CAG ATA GCA TGA AG -' Reverse: 5'- TAG GGC GGA TAG GTG GTA AT -' Forward: 5'- ACC CTT CAC CAA TGA CTC CTA TG -' Reverse: 5'- ATG ATG ACT GCA GCA AAT CGC -' 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-' Reverse: 5'- CTG CTG CCT TTG TTG CTC TTC CAA-' Forward: 5'- GAC TCT CTG CCT GGG AAT TT -' Reverse: 5'- GGA CGT ATC AAT GTC AGC AAA C -' Forward: 5'- CTC GGA GGG TTT CTT GTT TCT -' Reverse: 5'- CAG GAA TAG GGA CAA GAC AGA TG-' Forward: 5'- AGA GTG AGA CTC CAC CTC AA -' Reverse: 5'- CAG CCA GTT GCC CAT CTA TAA -' Forward: 5'- AGC CTC ATG AAG AGC CTT CCA Reverse: 5'- TCC GGA AGA AAC CCT TGC A Forward: 5'- CGT CAC TTC CAC GAG AGT TTA T Reverse: 5'- TCC CAC AGA ATG TTG TAG AGT TC