SUPPLEMENTARY DATA Supplementary Table 1. Clinical ... - Diabetes

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The antibodies for flow cytometry are listed. The antibodies were purchased from BD Bioscience (San Diego, CA), eBioscience or AbD Serotec. (Oxford, UK).

SUPPLEMENTARY DATA  

Supplementary Table 1. Clinical characteristics and metabolic parameters. 18 Japanese male patients were classified by BMI, according to the criteria of the Japanese Society for the Study of Obesity. Some patients were treated with anti-hypertensive medicines (BMI 25, 3/7) and oral antidiabetic medicines (BMI 25, 1/7). Data are shown as means  SE. *P < 0.01. All participants gave written informed consent. BMI 25 kg/m2 group (obese) 7 64 ± 3 26.6 ± 0.3 *

Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) White blood cell count (cells/mL) Creatinine (mg/dL) Fasting blood glucose (mg/dL)

126.0 ± 4.6 71.1 ± 3.8 6591 ± 449 0.84 ± 0.06 113 ± 11

131.7 ± 5.3 76.7 ± 5.5 6960 ± 755 0.83 ± 0.04 119 ± 8

 

Supplementary Table 2. Antibodies for flow cytometry. The antibodies for flow cytometry are listed. The antibodies were purchased from BD Bioscience (San Diego, CA), eBioscience or AbD Serotec (Oxford, UK). Antibody Anti-CD45R/B220 Anti-CD11c/Mac-1 Anti-CD11c Anti-CD8 Anti-CD19 Anti-CD45 Anti-CD11c Anti-CD11c Anti-CD3 Anti-Ly6G/Gr-1 Anti-F4/80 Anti-RP105 Anti-MD14 Anti-TLR4/MD-2 Anti-RP105 Anti-F4/80 Anti-CD206

Clone RA3-6B2 M1/70 HL3 53-6.7 1D3 30-F11 HL3 HL3 145-2C11 RB6-8C5 BM8 RP/14 RA3-6B2 MTS510 RP/14 BM8 MR5D3

Conjugate Biotin Biotin Biotin Biotin Biotin FITC PE APC Biotin Biotin FITC PE PE PE Biotin APC Alexa Fluor 647

Source BD Bioscience BD Bioscience BD Bioscience BD Bioscience BD Bioscience BD Bioscience BD Bioscience BD Bioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience eBioscience AbD Serotec

                  ©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA  

Supplementary Table 3. RT-qPCR primers. The primers were purchased from Applied Biosystems. Species Gene

Name

Assay ID

Mouse

Mm00446968_m1

RP105

hypoxanthine guanine phosphoribosyl transferase 1 CD180 antigen

MD-1

lymphocyte antigen 86

Mm00440240_m1

TLR4

toll-like receptor 4

Mm00445273_m1

MD-2

lymphocyte antigen 96

Mm00444223_m1

TNF-

tumor necrosis factor

Mm00443258_m1

MCP-1

chemokine (C-C motif) ligand 2

Mm00441243_g1

Human

Hprt

Mm00440240_m1

Adiponectin adiponectin, C1Q and collagen domain containing Arg1 arginase, liver

Mm00456425_m1

Mgl2

Mm00460844_m1

iNOS

macrophage galactose N-acetyl-galactosamine specific lectin 2 nitric oxide synthase 2, inducible

CD11c

integrin alpha X

Mm00498698_m1

F4/80

Mm00802530_m1

IKK-

EGF-like module containing, mucin-like, hormone receptor-like sequence 1 inhibitor of kappaB kinase epsilon

IL-6

interleukin 6

Mm00446191_m1

MyD88

Mm01351743_g1

TRIF

myeloid differentiation primary response gene 88 toll-like receptor adaptor molecule 1

TLR1

toll-like receptor 1

Mm00446095_m1

TLR2

toll-like receptor 2

Mm00442346_m1

TLR3

toll-like receptor 3

Mm01207403_m1

TLR5

toll-like receptor 5

Mm00546288_s1

TLR6

toll-like receptor 6

Mm02529782_s1

TLR7

toll-like receptor 7

Mm00446590_m1

TLR8

toll-like receptor 8

Mm01157262_m1

TLR9

toll-like receptor 9

Mm00446193_m1

HPRT

hypoxanthine phosphoribosyltransferase 1

Hs01003267_m1

RP105

CD180 molecule

Hs01069872_m1

MD-1

lymphocyte antigen 86

Hs00169454_m1

Mm01190441_g1

Mm01309898_m1

Mm00444862_m1

Mm00844508_s1

        ©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA  

Supplementary Figure 1. HFD treatment increases the expression of RP105 mRNA in the subcutaneous and retroperitoneal white adipose tissues. RT-qPCR of TLR4, MD-2, RP105, MD-1, TNF and MCP-1 mRNA in the subcutaneous and retroperitoneal adipose tissues from WT mice fed with ND or HFD for 12 weeks (n = 6 per group). Data are presented relative to the expression in ND mice, set as 1. *P < 0.05 versus ND. **P < 0.005 versus ND.

 

Supplementary Figure 2. HFD treatment increases the expression of RP105 mRNA in the liver, BAT and skeletal muscle. RT-qPCR of TLR4, MD-2, RP105, MD-1, MyD88 and TRIF mRNA in the liver, BAT, skeletal muscle, spleen and bone marrow from WT mice fed with ND or HFD for 12 weeks (n = 8 per group). Data are presented relative to the expression in ND mice, set as 1. *P < 0.05 versus ND.

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA   Supplementary Figure 3. MD-1 mRNA expression in human adipose tissues. (A) Linear regression

analysis of correlation between adipose MD-1 mRNA expression and BMI. (B) MD-1 mRNA expression of the human adipose tissues of nonobese (BMI 25 kg/m2, n = 7) subjects. Data are shown as means  SE. *P < 0.05.

Supplementary Figure 4. Expression of RP105, MD-1, TLR4 and MD-2 mRNA in CD45+ or CD45SVF cells from wild-type mice fed with ND or HFD. RT-qPCR of RP105, MD-1, TLR4 and MD-2 mRNA in CD45+ or CD45- SVF cells from WT mice fed with ND or HFD for 12 weeks (n = 7 per group). Data are shown as means  SE. *P < 0.05 versus ND.

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA  

Supplementary Figure 5. Expression of RP105/MD-1 and TLR4/MD-2 in spleen cells. (A) Flow cytometry analysis of RP105, MD-1 and TLR4/MD-2 expression on Mac-1+, CD19+ and CD3+ spleen cells from WT mice fed with ND. (B) RT-qPCR of RP105, MD-1, TLR4 and MD-2 mRNA in whole spleen cells, Mac-1+, CD19+ and CD3+ spleen cells from WT mice fed with ND. N.D., not detected. Similar results were obtained in three independent experiments.

Supplementary Figure 6. Increased expression of RP105 mRNA in the co-cultured cells is independent of TLR4 signaling. Peritoneal macrophages from WT or TLR4 KO mice were co-cultured with differentiated 3T3-L1 cells for 24 h. Then, expression of RP105 mRNA in control and co-cultured cells was measured by RT-qPCR (n =3 per group). Data are shown as means  SE. Similar results were obtained in three independent experiments.

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA  

Supplementary Figure 7. Representative photos and MRI images of WT, RP105 KO, MD-1 KO and TLR4 KO mice. The mice were fed with HFD or ND starting at 10 weeks of age for 12 weeks. (A) The photos are representative of 12 mice per each group. (B) The MRI images are representative of 12 mice fed with HFD per each group. The adiposity of mice was examined by a MRI scanning, MRmini SA (DS Pharma Biomedical Co., Ltd., Suita, Osaka).

Supplementary Figure 8. Locomotor activity and respiratory quotient (RQ) of WT, RP105 KO, MD-1 KO and TLR4 KO mice fed with HFD. Locomotor activity (A) and RQ (B) were measured for WT, RP105 KO, MD-1 KO and TLR4 KO mice fed with HFD for 12 weeks (n = 6 per group). Data are shown as means.

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1

SUPPLEMENTARY DATA  

Supplementary Figure 9. Inflammatory signaling protein expression in eWAT and SVF. Protein levels of phospho-JNK, JNK, IB, phospho-IKK and IKK were measured by western blotting with lysates from eWAT (A left panel, C) or SVF (A right panel, B) from WT, RP105 KO, MD-1 KO and TLR4 KO mice fed with ND or HFD for 12 weeks. Three mice in each group were used in A (left panel). In A (right panel), B and C, two and three mice were used in ND and HFD group, respectively. Actin was used as an internal loading control. Anti-phospho-JNK, anti-JNK and anti-IB were purchased from Cell Signaling. Anti-phospho-IKK was purchased from Young in frontier (Seoul, South Korea).

 

©2012 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1182/-/DC1