Procedures for RT-qPCR -...

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SUPPLEMENTARY DATA ©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1 Procedures for RT-qPCR: 1. Quantification and integrity measurement of RNA: Following a primary measurement using a Nanodrop 2000 spectrophotometer (Thermo, Wilmington, DE), the concentrations of RNA samples were adjusted to 200-300 ng/μl using RNase-free H 2 O. Subsequently the concentrations of RNA were determined triplicate using a Nanodrop 2000 spectrophotometer. The ratios of 260/280 nm and 260/230 nm in the RNA samples isolated from WAT and liver are 2.03-2.10 and 2.24-2.63, respectively. In addition, the integrity of RNA was confirmed using an Agilent 2100 Bioanalyzer (Palo Alto, CA) with an Agilent RNA 600 Nano Kit. The RNA Integrity Numbers of the RNA samples used in the present study are above 8.2. 2. RT-qPCR: Total RNA (500 ng) was reverse transcribed with MuLV reverse transcriptase and Oligod(T) primers in a 50 μl volume (Table S2). All the reagents for the reverse transcription (RT) were from Applied Biosystems (ABI, Foster City, CA). Resulting RT products were diluted 2 times using RNase-free H 2 O and applied in the following qPCR (Table S2). Each target mRNA in all the samples was measured triplicate. In each plate, three wells without template (with the same volume of DNase-free H 2 O) were used as negative controls. 3. Reference gene selection for target gene normalization: 18S has been reported as the most stable endogenous control under various experimental conditions, including adipocytes (1) and adipose tissues from obese and diabetic patients (2). To further confirm 18S is a proper reference gene in WAT of current study, the expression of 18S, β-actin (forward primer: 5’- GTATGACTCCACTCACGGCAAA-3’; reverse primer: 5’-GGTCTCGCTCCTGGAAGATG-3’) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh, forward primer: 5’- AGTATGACTCCACTCACGGCAAAT-3’; reverse primer: 5’-GTCTCGCTCCTGGAAGATGGT-3’) was determined using the protocol described above. Consistent with previous report (1; 2), 18S is the most stable endogenous control in WAT among the four experimental groups (Nrf2+/+:WT, Nrf2-/- :WT, Nrf2+/+:ob/ob, Nrf2-/-:ob/ob), with Cq values (mean ± SD) 14.68 ± 0.29, 14.24 ± 0.03, 14.50 ± 0.02 and 14.38 ± 0.16, respectively. There were no significant differences in relative target gene expression when normalized to undiluted 18S at 12.5 ng compared to diluted 18S at 12.5 pg, representing a 10 cycle difference in Cq value. In contrast, substantially increased levels of β-actin and Gapdh (with lower Cq values) were found in WAT of ob/ob mice compared to non-ob/ob mice. Thus, 18S was selected as the reference gene for target gene normalization in the present study. Preparation of tissue lysates of WAT for immunoblotting. WAT (1,000 mg) was homogenized in 500 μl 2× lysis buffer (Cell Signaling; #9803) with protease and phosphatase inhibitors (Sigma; P8340; P0044; P5726) and 1875 μl of a chloroform/methanol (1:2) mixture using a TissueLyser II. Following homogenization, delipidation of tissue lysates was performed by chloroform/methanol extraction (8). Briefly, homogenized tissue was transferred to 10 ml glass tubes and mixed sporadically while kept on ice for 10–15 min. Subsequently the homogenate was diluted with 625 μl of chloroform and 625 μl of water to change the water/chloroform/methanol ratio from 0.8:1:2 to 1.8:2:2 in the final organic solution. Following centrifugation (800g, 5 min, 4 °C), protein disk between lipid (lower) phase and aqueous (upper) phase was collected and dissolved in 1× lysis buffer. Resulting protein solution was sonicated for 10 seconds and used for protein quantification and further immunoblot analysis.

Transcript of Procedures for RT-qPCR -...

Page 1: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Procedures for RT-qPCR:

1. Quantification and integrity measurement of RNA: Following a primary measurement using a Nanodrop 2000 spectrophotometer (Thermo, Wilmington, DE), the concentrations of RNA samples were adjusted to 200-300 ng/μl using RNase-free H2O. Subsequently the concentrations of RNA were determined triplicate using a Nanodrop 2000 spectrophotometer. The ratios of 260/280 nm and 260/230 nm in the RNA samples isolated from WAT and liver are 2.03-2.10 and 2.24-2.63, respectively. In addition, the integrity of RNA was confirmed using an Agilent 2100 Bioanalyzer (Palo Alto, CA) with an Agilent RNA 600 Nano Kit. The RNA Integrity Numbers of the RNA samples used in the present study are above 8.2.

2. RT-qPCR: Total RNA (500 ng) was reverse transcribed with MuLV reverse transcriptase

and Oligod(T) primers in a 50 μl volume (Table S2). All the reagents for the reverse transcription (RT) were from Applied Biosystems (ABI, Foster City, CA). Resulting RT products were diluted 2 times using RNase-free H2O and applied in the following qPCR (Table S2). Each target mRNA in all the samples was measured triplicate. In each plate, three wells without template (with the same volume of DNase-free H2O) were used as negative controls.

3. Reference gene selection for target gene normalization: 18S has been reported as the most stable endogenous control under various experimental conditions, including adipocytes (1) and adipose tissues from obese and diabetic patients (2). To further confirm 18S is a proper reference gene in WAT of current study, the expression of 18S, β-actin (forward primer: 5’-GTATGACTCCACTCACGGCAAA-3’; reverse primer: 5’-GGTCTCGCTCCTGGAAGATG-3’) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh, forward primer: 5’-AGTATGACTCCACTCACGGCAAAT-3’; reverse primer: 5’-GTCTCGCTCCTGGAAGATGGT-3’) was determined using the protocol described above. Consistent with previous report (1; 2), 18S is the most stable endogenous control in WAT among the four experimental groups (Nrf2+/+:WT, Nrf2-/-:WT, Nrf2+/+:ob/ob, Nrf2-/-:ob/ob), with Cq values (mean ± SD) 14.68 ± 0.29, 14.24 ± 0.03, 14.50 ± 0.02 and 14.38 ± 0.16, respectively. There were no significant differences in relative target gene expression when normalized to undiluted 18S at 12.5 ng compared to diluted 18S at 12.5 pg, representing a 10 cycle difference in Cq value. In contrast, substantially increased levels of β-actin and Gapdh (with lower Cq values) were found in WAT of ob/ob mice compared to non-ob/ob mice. Thus, 18S was selected as the reference gene for target gene normalization in the present study. Preparation of tissue lysates of WAT for immunoblotting. WAT (1,000 mg) was homogenized in 500 μl 2× lysis buffer (Cell Signaling; #9803) with protease and phosphatase inhibitors (Sigma; P8340; P0044; P5726) and 1875 μl of a chloroform/methanol (1:2) mixture using a TissueLyser II. Following homogenization, delipidation of tissue lysates was performed by chloroform/methanol extraction (8). Briefly, homogenized tissue was transferred to 10 ml glass tubes and mixed sporadically while kept on ice for 10–15 min. Subsequently the homogenate was diluted with 625 μl of chloroform and 625 μl of water to change the water/chloroform/methanol ratio from 0.8:1:2 to 1.8:2:2 in the final organic solution. Following centrifugation (800g, 5 min, 4 °C), protein disk between lipid (lower) phase and aqueous (upper) phase was collected and dissolved in 1× lysis buffer. Resulting protein solution was sonicated for 10 seconds and used for protein quantification and further immunoblot analysis.

Page 2: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Table 1. Primer sequences for mouse genotyping

Supplementary Table 2. Reaction conditions for RT-qPCR

RTABI Reagents Volume (μl) RT conditionRNase-free H2O 9.25 25˚C 10 minMgCl2 (25 mM) 11 48˚C 60 minGeneAmp 10X PCR Buffer II 5 95˚C 5 mindNTP (10 mM) 10 4˚C ∞Oligd(T)16 (50 μM) 2.5Rnase inhibitor (20 U/μl) 1MuLV reverse transcriptase (5 U/μl) 1.25RNA (50 ng/μl) 10Total volume 50

qPCR Volume (μl) PCR condition RepeatsSYBR 3.5 50˚C 2 min 1Primer Mix (10 μM) 0.45 95˚C 10 min 1Dnase-free H2O 1.05 95˚C 0.15 min 40RT products 2.5 60˚C 1 min 40Total volume 7.5 4˚C ∞ 1

Primer name Sequence (5'-3') PCR products (bp)Nrf2-knockout:Nrf2-forward TGGACGGGACTATTGAAGGCTGNrf2-reverse CGCCTTTTCAGTAGATGGAGG 733 (WT)LacZ-reverse GCGGATTGACCGTAATGGGATAGG 500 (KO)Nrf2-LoxP:Nrf2-LoxP-forward CACAATGGTATGCCTGCTGTNrf2-LoxP-WT-reverse TCTGCACCAGAGTTCAAAGG 218 (WT)Nrf2-LoxP-KI-reverse AAGAGGGGGTTGGAAAGAGA 174 (KI)

Cre see The Jackson Laboratory protocolOb see The Jackson Laboratory protocol

Page 3: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

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©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Table 3. Primer sequences for RT-qPCR

Abbreviations Gene name Forward (5' - 3') Reverse (5' - 3') PCR efficiency (%)18S 18S ribosomal RNA CGAACGTCTGCCCTATCAACTT CCGGAATCGAACCCTGATT 101.5Acacα Acetyl-CoA carboxylase α CGGCAGTACCTGCGAGTAGAG GGGCGAATACACATTTGTCGTA 102.1Acacβ Acetyl-CoA carboxylase β GGGCCCTGGGAGACAAGA GGGTAAGGTTGGGATTTGCA 103.9Adfp Adipose differentiation-related protein GGTGATGGCAGGCGACAT CCATCGGACACTTCCTTAAAGG 92.8

Adipoq Adiponectin GCTCAGGATGCTACTGTTGCAA AACGTCATCTTCGGCATGACT 90.7Adpsn Adiposin GCTATCCCAGAATGCCTCGTT TTCCACTTCTTTGTCCTCGTATTG 92Akt1 Akt1 TGTCTCGAGAGCGTGTGTTCTC CAGACACAATCTCCGCACCAT 102.7Akt2 Akt2 ATGAAGATCCTGCGCAAGGA GCAGAACCCGGCTCTCTGT 90.25Mcp1 Monocyte chemotactic protein-1 CTGAAGCCAGCTCTCTCTTCCT CAGGCCCAGAAGCATGACA 101.2Cd36 Cluster of differentiation 36 CAGAGTTCGTTATCTAGCCAAGGAA CATTGGGCTGTACAAAAGACACA 96.9Cd68 Cluster of differentiation 68 CCCATCCCCACCTGTCTCT TGATGTAGGTCCTGTTTGAATCCA 94.1Cebpα CCAAT-enhancer-binding protein α CGCAAGAGCCGAGATAAAGC CGGTCATTGTCACTGGTCAACT 94.7Cebpβ CCAAT-enhancer-binding protein β AAGCTGAGCGACGAGTACAAGA GTCAGCTCCAGCACCTTGTG 94.2Cebpδ CCAAT-enhancer-binding protein δ GCCGTGCCCACCCTAGA CGCTTTGTGGTTGCTGTTGA 104.8F4/80 EGF-like moleculecontaining mucin-like hormone TCAGCCATGTGGGTAC AGTCA CACAGCAGGAAGGTGGCTATG 106.9Fabp4 Fatty acid binding protein 4 GCGTGGAATTCGATGAAATCA CCCGCCATCTAGGGTTATGA 105Fas Fatty acid synthase CCTGGATAGCATTCCGAACCT AGCACATCTCGAAGGCTACACA 100.5Gclc Glutamate—cysteine ligase catalytic subunit TGGCCACTATCTGCCCAATT GTCTGACACGTAGCCTCGGTAA 100.9Gclm Glutamate—cysteine ligase regulatory subunit ACATTGAAGCCCAGGATTGG CCCCTGCTCTTCACGATGAC 99.3Glut4 Glucose transporter type 4 CGCACTAGCTGAGCTGAAGGA AGGAGCTGGAGCAAGGACATT 105.1Gpat Glycerol-3-phosphate acyltransferase 1 AGGCTTCTAGGTCCCCTGCTA CCGCTGAAGTTGTGGACAAA 96.4Gpx1 Glutathione peroxidase 1 CGCTTTCGTACCATCGACATC GGGCCGCCTTAGGAGTTG 95.7Gpx2 Glutathione peroxidase 2 ACCGATCCCAAGCTCATCAT CAAAGTTCCAGGACACGTCTGA 107.2Gpx3 Glutathione peroxidase 3 ACAGGAGCCAGGCGAGAA CCACCTGGTCGAACATACTTGA 99.4Gpx4 Glutathione peroxidase 4 CCCGATATGCTGAGTGTGGTT CCTGCCTCCCAAACTGGTT 91.2Gsr Glutathione reductase TTGCGTGAATGTTGGATGTGT TTCCGAGTGCACTGCTGTGT 94.2Gss Glutathione synthetase TGCGGTGGTGCTACTGATTG CGGCACGCTGGTCAAATAT 98.6Ho1 Heme oxygenase 1 CCTCACTGGCAGGAAATCATC CCTCGTGGAGACGCTTTACATA 97.1Ikkβ Inhibitor of nuclear factor kappa-B kinase subunit AGCTGTCCTTACCCTGCTGAGT ATGGATGATTCTGTTTTCGTGAAG 100.7Il6 Interleukin 6 GCCCACCAAGAACGATAGTCA GAAGGCAACTGGATGGAAGTCT 95.5

IL10 Interleukin 10 GATGCCCCAGGCAGAGAA CACCCAGGGAATTCAAATGC 104.5Il1β Interleukin 1β GAAACCATGGCACATTCTGTTC AATAGGTAAGTGGTTGCCCATCA 93.2Infγ Interferon γ TTGGCTTTGCAGCTCTTCCT TGACTGTGCCGTGGCAGTA 100Irs1 Insulin receptor substrate 1 CCTCAGTCCCAACCATAACCA TCCGGCACCCTTGAGTGT 92.8

Nos2 Nitric oxide synthase 2 GCAAACCCAAGGTCTACGTTCA GAGCACGCTGAGTACCTCATTG 97.88Nox2 NADPH oxidase gp91phox subunit CAGGAGTTCCAAGATGCCTG GATTGGCCTGAGATTCATCC 94.4Nqo1 NAD(P)H dehydrogenase (quinone 1) TATCCTTCCGAGTCATCTCTAGCA TCTGCAGCTTCCAGCTTCTTG 95.4Nrf2 Nuclear factor E2-related factor 2 A GCTCGACAATGTTCTCCAGCTT 99.3

Pepck1 Phosphoenolpyruvate carboxykinase 1 CCACAGCTGCTGCAGAACA GGGTCGCATGGCAAAGG 104.3Pepck2 Phosphoenolpyruvate carboxykinase 2 GCAAACTCCCCAAGTATAAGAACTG GCTTTCTACCCGTGCCACAT 95.3Pgc1α Peroxisome proliferator-activated receptor γ coac CCGTAGGCCCAGGTACGA TGCGGTATTCATCCCTCTTGA 99.3Atgl Adipose triglyceride lipase AGACAGAGCTTTCTCCCAGTGAA CCCCGTGAAGCCCAACT 106

Pparγ1 Peroxisome proliferator-activated receptor γ1 GGGCTGAGGAGAAGTCACAC TGGTTCACCGCTTCTTTCA 91Pparγ2 Peroxisome proliferator-activated receptor γ2 TGCTGTTATGGGTGAAACTCTG CTGTGTCAACCATGGTAATTTCTT 90Prx1 Peroxiredoxin 1 GATCCCAAGCGCACCATT TAATAAAAAGGCCCCTGAAAGAGA 98.2Retn Resistin CACGTACCCACGGGATGAA GGACAAGGAAGAAAAGGAAAAGG 91

Socs3 Suppressor of cytokine signaling 3 TGGACCCATTCGGGAGTTC TCTGACCCTTTTGCTCCTTAAAGT 92.1Sod1 Superoxide dismutase 1 GTGATTGGGATTGCGCAGTA TGGTTTGAGGGTAGCAGATGAGT 100.1Sod2 Superoxide dismutase 2 TTAACGCGCAGATCATGCA GGTGGCGTTGAGATTGTTCA 100Sod3 Superoxide dismutase 3 CATGCAATCTGCAGGGTACAA AGAACCAAGCCGGTGATCTG 104Tcf7l1 transcription factor 7-like 1 CCAGCACACTTGTCCAACAAA AGCGGGTGCATGTGATGA 96Tcf7l2 transcription factor 7-like 2 TGCTGCTGGTGGGTGAAAA CTCGTCGTTAGCGCCTAGGT 95.8Tnfα Tumor necrosis factor α CAGCCGATGGGTTGTACCTT GGCAGCCTTGTCCCTTGA 91.6Txn Thioredoxin CCGCGGGAGACAAGCTT GGAATGGAAGAAGGGCTTGATC 108

Txnrd1 Thioredoxin reductase 1 CAATCTGAGCTGCCGAACAA GGGATCTTTGGAGCCATTCA 107.2Zfp423 Zinc-finger protein 423 CCGCTGTGTGGTCTGTATGC ATGTGAAAGGTGCCATGGATCT 106

Note: The specificity of primers has been validated using melting curve analysis. The PCR efficiency was determined using mRNA of WAT, liver, 3T3-L1 cells, RAW 264.7 cells and/or MIN6 cells and calculated from equation [Efficiency (%) = 100 × (10(-1/slope)-1)]. In addition, most of the primer sets have been tested and confirmed in mouse cells with specific gene knockdown, overexpression and/or exposure to chemical activators (3-7).

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SUPPLEMENTARY DATA

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Supplementary Figure 1. (A) Generation of a conditional allele, via a sequence replacement strategy to knock-out the Nrf2 gene. The construct contains loxP sites that flank exon 5, a 2.6 kb 5’ short arm of homology (containing exons 2, 3 & 4), a 9.8 kb 3’ long arm of homology, a Diphtheria Toxin A (DTA) cassette, and a Neomycin (Neo) cassette flanked by frt sites for selective deletion. The Neo element allows for positive selection in ES cells, while the DTA element permits negative selection in ES cells. After homologous recombination of the conditional knock-out construct, the PGK-Neo is excised via Flp-e electroporation. The Nrf2 gene has normal expression until Cre-mediated deletion of exon 5. This recombination creates a drastic premature stop, which renders the Nrf2 gene inactive. (B) Gene expression of Nrf2 in adipose tissue-specific Nrf2-knockout mice (Nrf2(f)-/-). Deletion of the floxed Nrf2 in adipose tissue was achieved by crossing homozygous Nrf2LoxP/LoxP with B6.Cg-Tg(Fabp4-cre)1Rev/J heterozygous mice (Cre-positive). Nrf2 expression was determined by real-time RT-PCR. Nrf2(f)+/+, Nrf2LoxP/LoxP and Cre-negative; Nrf2(f)-/-, Nrf2LoxP/LoxP and Cre-positive. n = 4-5 males (age = 14-15 weeks). *p < 0.05 vs. Nrf2(f)+/+.

Nrf2 wt

Targeting vector

HomologousRecombinant

Nrf2flx

Nrf2

=loxP =frt

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Short Arm PGK-NEOPGK-DTA Long Arm

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Ex1 Ex3Ex2 Ex4

PGK-NEO

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Ex1 Ex3Ex2 Ex4 Ex5

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Supplementary Figure 2. Ob/ob female mice with global Nrf2 deletion exhibit reduced body weight, aggravated insulin resistance and hyperglycemia. A: Body weight analysis of mice maintained on a chow diet. n = 6-10. *p < 0.05 vs. Nrf2+/+:ob/ob mice at the same age. B: Cumulative food consumption (FC). n = 5. *p < 0.05 vs. Nrf2+/+:ob/ob mice at the same age. C: Fasting blood glucose. n = 16-30. *p < 0.05 vs. non-ob/ob mice with the same Nrf2 genotype; #p < 0.05 vs. Nrf2+/+:ob/ob mice. D: Fasting plasma insulin. n = 3-8. E: Homeostatic model assessment for insulin resistance (HOMA-IR). n = 3-8. F: Intraperitoneal glucose tolerance test. Mice were challenged with 0.5 mg of glucose/g body weight. n = 10-11. &p < 0.05 vs. Nrf2+/+:ob/ob mice with the same treatment. G: Intraperitoneal insulin tolerance test. Mice were challenged with insulin at 0.75 and 4 U/g of BW in non-ob/ob and ob/ob mice, respectively. n = 9-11.

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SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 3. Nrf2-/-:ob/ob female mice show reduced WAT mass and mild hepatic steatosis but trended increased plasma triglycerides. A: Weight of WAT. Retroperitoneal and gonadal depots were measured. n = 11-15. Animal age is 8-15 wks. B: Levels of triglycerides in liver. n = 7. C: Levels of triglycerides in plasma. n = 6. D: Levels of triglycerides in skeletal muscle. n = 5-6. Values in A-D are mean ± SD. *p < 0.05 vs. non-ob/ob mice with the same Nrf2 genotype; #p < 0.05 vs. Nrf2+/+:ob/ob mice.

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Supplementary Figure 4. Representative images of H&E stained adipose tissues (20×). S-WAT, subcutaneous WAT; E-WAT, epididymal WAT. BAT, brown adipose tissue. Animal age is 12-15 wks.

Nrf2+/+:WT

S-WAT

E-WAT

BAT

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Supplementary Figure 5. Plasma levels of free glycerol and free fatty acids in mice. Values are mean ± SD. n = 6 male or female mice

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Supplementary Figure 6. Representative images of H&E stained adipose tissues (20×) in male mice. E-WAT, epididymal WAT; BAT, brown adipose tissue. Animal age is 8-12 wks.

Nrf2(f)+/+:ob/ob Nrf2(f)-/-:ob/ob

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E-WAT

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Supplementary Figure 7. mRNA expression of antioxidant enzymes in liver. n = 3-6 males. Animal age = 8-10 wks. Values are mean ± SD. *p < 0.05 vs. Nrf2+/+:WT. #p < 0.05 vs. Nrf2+/+:ob/ob mice. The number in brackets following each gene name is the Cq value of that gene in Nrf2+/+:WT. The average Cq value of reference gene 18S is 14.

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300Gpx4 (21)

0100200300400

Txnrd1 (31)

050

100150200

Prx1 (18)

050

100150200

Txn (19)

*#

*

*

050

100150200

0100200300400

*#

0

200

400

600

0

200

400

600

* *#

Gclc (23) Gclm (19) Ho1 (27) Nqo1 (26)

Gss (23)Sod2 (20) Sod3 (27)Sod1 (17)

mR

NA

expr

essi

on in

live

r (%

of N

rf2+/

+:W

T)

Page 10: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 8. Expression of antioxidant genes in epididymal WAT. n = 3-6 males. Animal age is 8-10 wks. Values are mean ± SEM. *p < 0.05 vs. Nrf2+/+:WT. The number in brackets following each gene name is the Cq value of that gene in Nrf2+/+:WT. The average Cq value of reference gene 18S is 14.

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

0

50

100

150Gpx1 (16)

0100200300400

0

50

100

150

mR

NA

of W

AT

(% o

fNrf2

+/+:

WT) Sod1 (16) Sod3 (22)

0

200

400

600Sod2 (21)

**

0

100

200

300

0

100

200

300

mR

NA

of W

AT

(% o

fNrf2

+/+:

WT) Gpx2 (27) Gpx4 (19)

0

50

100

150Gpx3 (17)

* *0

50

100

150

050

100150200

Gss (18)

050

100150200

mR

NA

of W

AT

(% o

fNrf2

+/+:

WT)

0200400600800

0

100

200

300

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Txn (18) Txnrd1 (32) Prx1 (18)

*Gsr (24)

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SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 9. GSH and GSSG levels in whole blood and plasma in female mice. n = 8-18. Animal age = 8-15 wks. Values are mean ± SD. *p < 0.05 vs. Nrf2+/+:WT; #p < 0.05 vs. Nrf2+/+:ob/ob mice.

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

b

Nrf2

-/-:W

T

Nrf2

-/-:o

b/ob

0

10

20

30

0

4

8

12

16

*

*

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

b

Nrf2

-/-:W

T

Nrf2

-/-:o

b/ob

0

500

1000

1500

2000

0

2

4

6

* #

GS

SG

(μM

)G

SH

(μM

)Whole blood Plasma

Page 12: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 10. mRNA expression of antioxidant and inflammatory response genes in epididymal WAT of Nrf2(f)-/-:ob/ob mice. n = 3-4 males. Animal age is 8-10 wks. Values are mean ± SD. *p < 0.05 vs. Nrf2(f)+/+:ob/ob. The number in brackets following each gene name is the Cq value of that gene in Nrf2(f)+/+:WT. The average Cq value of reference gene 18S is 14.

0

50

100

150 Gclc (23)

0

50

100

150 Nqo1 (25)

0

50

100

150 Cd68 (18)

0

100

200

300 Il6 (24)

0

150

300

450

mR

NA

leve

lsin

WAT

(% o

f Nrf2

(f)+/

+:ob

/ob)

Infγ (29)

0

50

100

150

200 Il10 (25)

0

50

100

150

200F4/80 (21)

0

100

200

300 Il1β (22)

#

0

100

200

300 Tnfα (25)

0

50

100

150 Ikkβ (25)Mcp1 (23)

0

50

100

150

200 Nos2 (30)

#

0

50

100

150 Gclm (19)

0

50

100

150 Ho1 (23)

#

0

100

200

300Socs3 (23)

0

50

100

150

0

50

100

150

200 Nox2 (21)

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SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 11. Adipogenic gene expression in epididymal WAT. n = 3-6 males. Animal age is 8-10 wks. Values are mean ± SD. *, p < 0.05 vs. Nrf2+/+:WT; #p < 0.05 vs. Nrf2+/+:ob/ob mice. The number in brackets following each gene name is the Cq value of that gene in Nrf2+/+:WT. The average Cq value of reference gene 18S is 14.

050

100150200250

0100200300400500

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Tcf7l1 (28)

050

100150200250

050

100150200

0100200300400

Cebpα (21) Cebpδ (23)Cebpβ (25)

0

200

400

600

0

500

1000

1500

050

100150200250

Fas (27) Adfp (19) Cd36 (17)

050

100150200

Glut4 (25)

0

50

100

150Fabp4 (14)

0

100

200

300

050

100150200

*

Zfp423 (26) Pparγ2 (24)Pparγ1 (24)

mR

NA

leve

ls in

WAT

(% o

f Nrf2

+/+:

WT)

*

Page 14: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 12. mRNA expression profile in epididymal WAT. n = 3-6 males. Animal age is 8-10 wks. Values are mean ± SD. *, p < 0.05 vs. Nrf2+/+:WT. The number in brackets following each gene name is the Cq value of that gene in Nrf2+/+:WT. The average Cq value of reference gene 18S is 14.

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

Nrf2

+/+:

WT

Nrf2

+/+:

ob/o

bN

rf2-/-

:WT

Nrf2

-/-:o

b/ob

0

50

100

150

050

100150200250

Gpat (27)

050

100150200

Acacβ (25)

0500

100015002000

Atgl (24)

*

050

100150200

Adipoq (17)

0

50

100

150 Retn (17)

0

50

100

150

* *

Pepck1 (18) Pepck2 (26)

0

50

100

150Akt2 (22)

050

100150200

Irs1 (23)

050

100150200

0

50

100

150Adipsin (15)

* *

Acacα (27)

mR

NA

leve

ls in

WAT

(% o

f Nrf2

+/+:

WT)

Akt1 (26)

050

100150200

Page 15: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

Supplementary Figure 13. mRNA expression of adipogenic genes in epididymal WAT. n = 3-4 males. Animal age is 8-10 wks. Values are mean ± SD. # p < 0.05 vs. Nrf2(f)+/+:ob/ob. The number in brackets following each gene name is the Cq value of that gene in Nrf2(f)+/+:WT. The average Cq value of reference gene 18S is 14

0

50

100

150

mR

NA

leve

lin W

AT(%

of N

rf2(f)

+/+:

ob/o

b) Pparγ1 (22)

0

50

100

150

200Pparγ2 (21)

0

50

100

150Cebpα (19)

0

50

100

150Cebpβ (22)

0

50

100

150

200

mR

NA

leve

lin W

AT(%

of N

rf2(f)

+/+:

ob/o

b) Cebpδ (20)

0

50

100

150Tcf7l1 (27)

0

50

100

150Zfp423 (22)

0

50

100

150Cd36 (15)

0

50

100

150

200

mR

NA

leve

lin W

AT(%

of N

rf2(f)

+/+:

ob/o

b) Fas (24)

0

50

100

150 Glut4 (23)

0

50

100

150Adfp (16)

0

50

100

150Fabp4 (13)

#

Page 16: Procedures for RT-qPCR - Diabetesdiabetes.diabetesjournals.org/content/suppl/2012/12/12/db12-0584... · Procedures for RT-qPCR: 1. ... (Sigma; P8340; P0044; P5726) and 1875 μl of

SUPPLEMENTARY DATA

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0584/-/DC1

References:

1. Ferguson BS, Nam H, Hopkins RG, Morrison RF: Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes. PLoS One 2010;5:e15208 2. Catalan V, Gomez-Ambrosi J, Rotellar F, Silva C, Rodriguez A, Salvador J, Gil MJ, Cienfuegos JA, Fruhbeck G: Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus. Horm Metab Res 2007;39:495-500 3. Pi J, Zhang Q, Woods CG, Wong V, Collins S, Andersen ME: Activation of Nrf2-mediated oxidative stress response in macrophages by hypochlorous acid. Toxicol Appl Pharmacol 2008;226:236-243 4. Woods CG, Fu J, Xue P, Hou Y, Pluta LJ, Yang L, Zhang Q, Thomas RS, Andersen ME, Pi J: Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages. Toxicol Appl Pharmacol 2009;238:27-36 5. Hou Y, Xue P, Bai Y, Liu D, Woods CG, Yarborough K, Fu J, Zhang Q, Sun G, Collins S, Chan JY, Yamamoto M, Andersen ME, Pi J: Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein beta during adipogenesis. Free Radic Biol Med 2012;52:462-472 6. Xue P, Hou Y, Zhang Q, Woods CG, Yarborough K, Liu H, Sun G, Andersen ME, Pi J: Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: involvement of the adaptive antioxidant response. Biochem Biophys Res Commun 2011;407:360-365 7. Pi J, Leung L, Xue P, Wang W, Hou Y, Liu D, Yehuda-Shnaidman E, Lee C, Lau J, Kurtz TW, Chan JY: Deficiency in the nuclear factor E2-related factor-2 transcription factor results in impaired adipogenesis and protects against diet-induced obesity. J Biol Chem 2010;285:9292-9300 8. Sajic T, Hopfgartner G, Szanto I, Varesio E: Comparison of three detergent-free protein extraction protocols for white adipose tissue. Anal Biochem 2011;415:215-217