SUPPLEMENTARY DATA. Supplementary Table 1. Sequences of the primers and TaqMan probes

Transcription

1 Supplementary Table 1. Sequences of the primers and TaqMan probes Gene (GenBank accession) Oligonucleotide sequence (5-3 ) ARG1 (NM_000045) TACTAGGAAGAAAGAAAAGGCCAATTC GTAGCCCTGTTTTGTAGATTTCTTCTGT TaqMan Probe FAM-ACCCATCTTTCACACCAGCTACTGGCACA-TAMRA CCL2 (NM_002982) GCTCATAGCAGCCACCTTCATT TCTGCACTGAGATCTTCCTATTGGT TaqMan Probe FAM-TCGCTCAGCCAGATGCAATCAATGC-TAMRA CHI3L1 (NM_001276) GGACGGAGAGACAAACAGCATT CCTGGCTGGGCTTCCTTTAT TaqMan Probe FAM-CCACCCTAATCAAGGAAATGAAGGCC-TAMRA HIF1A (NM_ ) AACTAGCCGAGGAAGAACTATGAACA TACCCACACTGAGGTTGGTTACTGT TaqMan Probe FAM-AACATGGAAGGTATTGCACTGCACAGGC-TAMRA IL1A (NM_000575) GTTCTGAAGAAGAGACGGTTGAGTTT AAGTTGTATTTCACATTGCTCAGGAA TaqMan Probe FAM-CATCGCCAATGACTCAGAGGAAGAAATCA-TAMRA IL1B (NM_000576) CAGTGGCAATGAGGATGACTTG GTAGTGGTGGTCGGAGATTCGTA TaqMan Probe FAM-TGGCCCTAAACAGATGAAGTGCTCCTTCC-TAMRA IL4 (NM_000589) GCCTCCAAGAACACAACTGAGAA TGTCGAGCCGTTTCAGGAAT TaqMan Probe FAM-CTGCGACTGTGCTCCGGCAGTTCTA-TAMRA IL6 (NM_000600) GCCCTGAGAAAGGAGACATGTAAC ATCCATCTTTTTCAGCCATCTTTG TaqMan Probe FAM-AGGCACTGGCAGAAAACAACCTGAACC-TAMRA IL10 (NM_000572) ACATCAAGGCGCATGTGAACT TGGCTTTGTAGATGCCTTTCTCTT TaqMan Probe FAM-CGCTGTCATCGATTTCTTCCCTGTGAA-TAMRA IL13 (NM_002188) CTCATTGAGGAGCTGGTCAACAT CTGTCAGGTTGATGCTCCATACC TaqMan Probe FAM-AACCAGAAGGCTCCGCTCTGCAATG-TAMRA IL32A (NM_ ) GAGACAGTGGCGGCTTATTATGA GGCACCGTAATCCATCTCTTTCT TaqMan Probe FAM-CAGCACCCAGAGCTCACTCCTCTACTTGAA-TAMRA MMP2 (NM_004530) CCATTTTGATGACGATGAGCTATG GTTGTACTCCTTGCCATTGAACAA TaqMan Probe FAM-CTTGGGAGAAGGCCAAGTGGTCCGT-TAMRA MMP9 (NM_004994) GCCCGGACCAAGGATACAGT CCCCTCAGTGAAGCGGTACA TaqMan Probe FAM-ACGCGCTGGGCTTAGATCATTCCTCA-TAMRA MRC1 (NM_002438) TACAAAAAGGACAAACACCAAAACC

2 TTGTAAATAACCCACCCATCTTCAG TaqMan Probe FAM-CAACACCAGCTCCTCAAGACAATCCACC-TAMRA NOD2 (NM_ ) TTCAGGAATTACCAGTCCCATTG GGTCCTCAGCTTGGCCATATACT TaqMan Probe FAM-CCCTGCCTTTGGAAGCTGCCACA-TAMRA PPARG (NM_005037) GGGATGTCTCATAATGCCATCAG CCGCCAACAGCTTCTCCTT TaqMan Probe FAM-TTTGGGCGGATGCCACAGGC-TAMRA PTGS2 (NM_000963) GCTCAGCCATACAGCAAATCCT TGGTAAATCCCACACTCATACATA TaqMan Probe FAM-TGGTCCCACCCATGTCAAAACCGAGG-TAMRA SPP1 (NM_000582) CATCCAGTACCCTGATGCTACAGA GGCCTTGTATGCACCATTCAA TaqMan Probe FAM-ACATCACCTCACACATGGAAAGCGAGGA-TAMRA TGFB (NM_000660) GCCCAGCATCTGCAAAGC TCCTTGCGGAAGTCAATGTACA TaqMan Probe FAM-CACCAACTATTGCTTCAGCTCCACGGA-TAMRA TLR4 (NM_003266) CTGCGTGGAGGTGGTTCCTA CAGGTCCAGGTTCTTGGTTGAG TaqMan Probe FAM-TTTCTACAAAATCCCCGACAACCTCCCCT-TAMRA TNC (NM_002160) AGGCGATCCCAGACAGTCAGT TCCAGCTGACAGTAGCCGAATT TaqMan Probe FAM-TCCAGCTGACAGTAGCCGAATT-TAMRA TNF (NM_000594) CCCCAGGGACCTCTCTCTAATC ACATGGGCTACAGGCTTGTCA TaqMan Probe FAM-CCTCTGGCCCAGGCAGTCAGATCAT-TAMRA TRIB1 (NM_025195) CTGCGCTGCAAGGTGTTTC TGGTTTCCCCAAGGATCACTT TaqMan Probe FAM-ACAAAATCAGGCCTTACATCCAGCTGCC-TAMRA VEGFA (NM_ ) CAGCACAACAAATGTGAATGCA ACACGTCTGCGGATCTTGTACA TaqMan Probe FAM-AATCCCTGTGGGCCTTGCTCAGAGC-TAMRA ARG1, arginase-1; CCL2, monocyte chemoattractant protein-1; CHI3L1, chitinase 3-like 1 (YKL-40); HIF1A, hypoxia inducible factor 1 ; IL, interleukin; MMP, matrix metalloproteinase; MRC1, mannose receptor, C type 1; NOD2, nucleotide binding oligomerization domain containing 2; PPARG, peroxisome proliferator-activated receptor ; PTGS2, prostaglandin-endoperoxide synthase 2 (COX-2); SPP1, secreted phosphoprotein 1 (osteopontin); TGFB1, transforming growth factor ; TLR4, toll-like receptor 4; TNC, tenascin C; TNF, tumour necrosis factor- ; TRIB1 tribbles pseudokinase 1; VEGFA, vascular endothelial growth factor.

3 Supplementary Table 2. Effects of weight loss in obese patients after Roux-en-Y gastric bypass (RYGB) or following a conventional dietetic intervention. RYGB Conventional diet Before WL After WL Before WL After WL n Age (years) Body weight (kg) *** *** BMI (kg/m 2 ) *** *** Body fat (%) *** *** Waist (cm) *** *** WHR *** ** Fasting glucose (mg/dl) *** Fasting insulin ( U/mL) *** ** Triglycerides (mg/dl) *** *** Cholesterol (mg/dl) *** *** LDL-cholesterol (mg/dl) *** ** HDL-cholesterol (mg/dl) *** Leptin (ng/ml) *** * CRP (mg/l) *** Fibrinogen (mg/dl) Leucocyte (x10 9 /L) *** Neutrophils (%) *** Lymphocytes (%) *** Monocytes (%) Eosinophils (%) Basophils (%) ALT (UI/L) *** AST (UI/L) ** -GT (UI/L) * *** ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; CRP, C-reactive protein; -GT, -glutamyltransferase; RYGB, Roux-en-Y gastric bypass; WHR, waist to hip ratio; WL, weight loss. Data are mean SEM. Differences between groups were analyzed by two-tailed paired t-tests. * P<0.05, ** P<0.01 and *** P< vs before WL. CRP levels were logarithmically transformed for statistical analysis due to their non-normal distribution.

4 Supplementary Figure 1. mrna expression levels of IL32 in human visceral adipocytes after transfection with two pairs of sirnas (s17656 and s17657) at 100 pm sirna IL32/2x10 5 cells for 24 h. Gene expression levels in scrambled sirna cells (CTL) were assumed to be 1. Values are the mean SEM (n=6 per group). Differences between groups were analyzed by unpaired two-tailed Student s t tests. * P<0.05 and ** P<0.01 vs CTL.

5 Supplementary Figure 2. Localization of IL32 in human visceral adipose tissue by histological and gene expression analyses. (A) Real-time PCR analysis of IL32 transcript levels in visceral (VAT) and subcutaneous (SAT) adipose tissue of lean and obese subjects. Bars represent the mean SEM of the ratio between gene expression to 18S rrna. The expression level in VAT was assumed to be 1. Differences between groups were analysed by unpaired two-tailed Student s t tests (n=30 per group, * P<0.05 vs VAT). Immunohistochemical (B) and immunofluorescence (C) detection of IL-32 in histological sections of human visceral adipose tissue. Representative images of at least 3 separate experiments are shown. (D) Comparison of IL32 gene expression in adipocytes and SVFCs isolated from VAT of obese patients. Bars represent the mean SEM of the ratio between the gene expression to 18S rrna. The expression level in adipocytes was assumed to be 1 (adipocytes: n = 9; SVFCs: n = 11). Statistical differences were assessed by two-tailed unpaired Student s t-test. *** P<0.001 vs adipocytes.