Supplementary information FTO intronic SNP strongly influences human neck adipocyte browning determined by tissue and PPARγ specific regulation: a transcriptome analysis Beáta B. Tóth, Rini Arianti, Abhirup Shaw, Attila Vámos, Zoltán Veréb, Szilárd Póliska, Ferenc Győry, Zsolt Bacso, László Fésüs*, Endre Kristóf* Supplementary Figure 1. A B C Supplementary Figure 1. Functional features of the primary SC and DN adipocytes differentiated for two weeks to white or brown. (A) Basal, cAMP stimulated and oligomycin inhibited oxygen consumption (OC) levels (as compared to basal OCR of SC white adipocytes) n=4. (B) Basal and cAMP-stimulated extracellular acidification levels (as compared to basal ECAR of SC white adipocytes) n=4. (C) The proportion of creatine kinase futile cycle related OC at basal and cAMP-stimulated n=4. SC: Subcutaneous, DN: Deep-neck, W: white differentiation protocol, B: brown differentiation protocol. Statistics: In comparison of two groups two-tailed paired Student’s t-test was used. *p<0.05. In multi-factor comparision we used two- way ANOVA and post hoc Tukey’s test. *p<0.05. **p<0.01. n=4 Supplementary Figure 2. Expression of ProFAT brown markers Z-score by donor -2.7 2.7 Supplementary Figure 2. Expression profile of ProFAT marker genes. Heat map shows the expression profile of browning (44) and white (6) characteristic marker genes from ProFAT database in SC and DN adipose progenitors and differentiated samples. Supplementary Figure 3. UCP1 CPT1B EHHADH AKAP1 SOD2 ACSL5 ZIC1 PRDM16 CKMT1A CKMT1B CIDEA PM20D1 Supplementary Figure 3. Gene-expression of key browning marker genes based on RNAseq data. SC: Subcutaneous DN: Deep-neck. W: white differentiation protocol; B: brown differentiation protocol; Statistics: Deseq R data. *p<0.05. n=9 donors. 4 samples/donor. Supplementary Figure 4. A B C D Supplementary Figure 4. Interactome maps of group 1-4 genes (A) Interactome map of group 1 genes. (B) Interactome map of group 2 genes (C) Interactome map of group 3 genes (D) Interactome map of group 4 genes. The interaction network was determined by STRING (https://string-db.org) and constructed by using Gephi 0.9.2 (https://gephi.org). Size of the nodes reflect the fold change of DN/SC (A and B) and SC/DN (C and D). Edges represent protein- protein interactions and the size of the edges indicate the confidence of interaction or the strenght of data support. The clustering was performed based on reactome (reactome.org) and KEGG Mapper (www.kegg.jp) Supplementary Figure 5. A B Brown markers from ProFAT database Brown markers from BATLAS database C D 81 DEGs encoding mitochondrial protein based on the presence 1036 mitochondrial protein coding genes based on the of FTO obesity-risk allele presence of FTO obesity-risk allele Supplementary Figure 5. FTO obesity-risk allele presence based analyses of gene-expression profiles. (A) Expression profile of ProFAT marker genes. Heatmap shows the expression profile of browning (44) and white (6) characteristic marker genes from ProFAT database in SC and DN adipose progenitors and differentiated samples; samples was hierarchically clustered based on pearson correlation. n=9 (B) Expression profile of adipocyte specific BATLAS marker genes. Heatmap shows the expression profile of browning (98) and white (21) characteristic marker genes from BATLAS database in SC and DN adipose progenitors and differentiated samples, samples was hierarchically clustered based on pearson correlation. To indentify donor differences z-score was calculated by considering all donors. n=9. (C) Expression profile of the 81 genes encoding mitochondrial proteins, which expressed differently according FTO obesity- risk allele presence. (D) Expression profiles of mitochondrial proteins encoding genes based on Human MitoCarta 2.0 database. Heat map shows the expression profiles of 1038 genes encoding mitochondrial proteins based on the presence of the FTO obesity-risk allele C/C in SC and DN adipose progenitors and differentiated samples. z-score was calculated by all samples to identify donor differences. n = 6 donors. SC Subcutaneous; DN: Deep-neck; W white differentiation protocol; B brown differentiation protocol. Numbers 1-9 represents donors, * outlier from the cluster. Supplementary Figure 6. A B Supplementary Figure 6. Number of differentially expressed genes in differentiated adipocytes based on tissue origin, differentiation protocol and the presence of the FTO obesity-risk allele. (A) Venn diagram shows significantly higher expressed genes number in samples expected higher thermogenic activity, and among of these genes several were lower expressed in FTO obesity-risk genotype samples (B) shows significantly lower expressed genes number in samples expected higher thermogenic activity, and among of these genes many expressed higher in FTO obesity-risk genotype samples. SC: subcutaneous; DN: deep-neck. Supplementary Table 1. Percentage of positive cells Percentage of positive cells Indicated cell type or component CD markers (SC PA) (DN PA) CD34 0.01±0.01 56.31±48.93 CD45 0.00±0.00 0.00±0.00 Hematopoietic/ Monocyte markers CD47 96.24±1.36 94.36±1.72 CD338 (ABCG2) 27.32±1.36 35.61±32.46 HLA-DR 30.22±52.34 28.46±49.29 CD31 (PECAM) 0.00±0.00 3.43±5.94 Endothelial markers CD54 (ICAM-1) 37.94±39.11 32.79±56.73 CD73 96.04±1.84 94.49±5.59 MSC/Fibroblast markers CD90 (Thy-1) 94.62±1.20 87.78±6.23 CD105 (Endoglin) 56.81±49.23 20.53±35.56 CD29 (Integrin ß1) 87.93±15.62 97.27±3.23 CD36 76.40±4.32 79.22±7.78 CD44 (H-CAM.Hermes) 85.31±11.82 60.29±48.31 Integrins and CAMs CD49a (Integrin α1) 50.50±45.76 68.26±4.89 CD49d (Integrin α4) 27.67±47.92 19.12±33.11 CD146 (MCAM) 4.13±6.66 0.96±1.67 CD325 81.75±13.17 90.92±6.46 Supplementary Table 1. Analysis of the surface antigen patterns of SC and DN preadipocytes. Expression of 17 markers was determined in undifferentiated preadipocytes of 3 independent donors with T/C genotype for the rs1421085 locus in the FTO gene by flow cytometry. Four groups of markers were tested: hematopoietic/monocyte, endothelial, MSC/fibroblast markers, integrins and CAMs. The numbers represent the percentage of positive cells ±SD. SC: Subcutaneous, DN: Deep-neck, PA: preadipocyte. Supplementary Table 2. GENE ID ENCODED PROTEIN ROLE IN ADIPOGENESIS IGF1 Insulin-like growth factor I Differentiation signal SOX9 Transcription factor SOX-9 Differentiation signal PPARG Peroxisome proliferator-activated receptor gamma Adipogenic transcription factor STAT3 Signal transducer and activator of transcription 3 Differentiation signal CEBPA CCAAT/enhancer-binding protein alpha Adipogenic transcription factor SREBF1 Sterol regulatory element-binding protein 1 Lipogenic gene regulators SLC2A4 Glucose transporter member 4 Adipocyte protein FABP4 Fatty acid-binding protein. adipocyte Adipocyte protein LPL Lipoprotein lipase Adipocyte protein AGPAT2 1-acylglycerol-3-phosphate O-acyltransferase 2 Adipocyte protein PLIN1 Perilipin-1 Adipocyte protein ADIPOQ Adiponectin Adipocyte protein LEP Leptin Adipocyte protein STAT5A Signal transducer and activator of transcription 5 Adipogenesis regulator EBF1 Transcription factor COE1/Early B-factor 1 Adipogenesis regulator CIDEC Cell death inducing DFFA like effector C Lipid droplet formation PPARGC1A Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Transcriptional co-factor PPARGC1B Peroxisome proliferator-activated receptor gamma coactivator 1-beta Transcriptional co-factor Supplementary Table 2. List of general adipocyte marker genes, the encoded proteins and their role in adipogenesis Supplementary Table 3. FC pValue DNW_SCW/DNB_SCB FC DNB_DNW/SCB_SCW pValue DNB_DNW/SCB_SCW Genes Encoded Protein DNW_SCW/DNB_SCB GROUP 1 AC007938.2 -/2.6 -/0.02 2.6/- 0.03/- AC009126.1 -/1.8 -/0.009 2.3/- 0.0003/- AC034102.6 -/2.3 -/0.01 2.3/- 0.02/- AL606534.4 3.6/3.2 0.003/0.005 2.7/2.9 0.04/0.03 Aryl hydrocarbon receptor nuclear translocator-like protein ARNTL2 2.1/2.3 0.001/4.2E-05 2.2/1.9 9.5E-04/1.5E-02 2 Lactosylceramide 1,3-N-acetyl-beta-D- B3GNT5 2.2/1.9 0.009/0.04 2.2/2.6 0.009/0.002 glucosaminyltransferase CIDEA Cell death activator CIDE-A 3.6/- 0.03/- 3.8/7.9 0.02/0.0001 CKMT1B Creatine kinase 1B 3.1/- -/2.6 -/0.04 CPA2 Carboxypeptidase A2 2.9/- 0.01/- 3.5/4.9 0.0004/8.5E-05 CPT1B Carnitine O-palmitoyltransferase 1, muscle isoform 1.8/2.1 0.015/0.0001 2.3/2.0 4.6E-05/8.2E-03 DAZL Deleted in azoospermia-like -/3.9 -/0.003 3.6/- 0.01/- DRP2 Dystrophin-related protein 2 -/2.3 -/0.007 2.4/- 0.005/- FAM151A Protein FAM151A 4.5/2.9 1.2E-05/0.002 2.4/3.7 0.03/0.0007 FAM189A2 Protein FAM189A2 -/2.6 -/0.006 2.3/- 0.03/- GPR160 Probable G-protein coupled receptor 160 -/2.5 -/0.0003 2.4/- 0.001/- KCNIP2 Kv channel-interacting protein 2 /2.4 /0.02 2.7/2.3 0.008/0.03 KCNIP2-AS1 -/2.5 -/0.01 2.5/- 0.01/- LINC01347 3.1/2.7 0.0007/0.01 2.6 0.05 LINC02458 -/2.6 -/0.03 2.9/- 0.01/- OASL 2'-5'-oligoadenylate synthase-like protein 4.1/- 0.006/- 5.2/11.0 0.001/1.0E-06 PCDH19 2.7/4.7 0.05/0.0001 4.4/- 0.0005/- PM20D1 N-fatty-acyl-amino acid synthase 4.5/4.2 0.003/0.006 7.0/7.5 9.8E-05/5.8E-05 RASD1 Dexamethasone-induced Ras-related protein 1 2.04/- 0.02/- 2.6/3.4 1.7E-03/2.6E-05 RASSF6 -/2.7 -/0.03 2.9/- 0.04/- Ras association domain-containing protein 6 RGL3 Ral guanine nucleotide dissociation stimulator-like 3 2,4/- 0.01/- 2.3/3.1 2.8E-02/1.5E-03 RXRG Retinoic acid receptor RXR-gamma 2.7/- 0.02/- -/2.8 -/0.02 SCN4A Sodium channel protein type 4 subunit alpha 3.7/- 2.3E-05/- 2.8/6.3 1.3E-03/5.7E-09 SLC19A3 Thiamine transporter 2 1.9/- 0.028/- 2.0/2.1 4.6E-02/2.2E-02 SLC7A10