Reverse primer sequence

1

Supplementary table 1. Primer sequences used for RT-qPCR.

Genes

Full name

Fgf21 fibroblast growth factor 21 Ppargc1a peroxisome proliferative activated receptor, gamma, coactivator 1 alpha Ucp1 uncoupling protein 1 Cck cholecystokinin Tph2 tryptophan hydroxylase 2 Acadl acyl-Coenzyme A dehydrogenase, long-chain Cpt1a carnitine palmitoyltransferase 1a Fasn fatty acid synthase Gys2 glycogen synthase 2 Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 S100a8 S100 calcium binding protein A8 (calgranulin A) Reference genes B2m beta-2 microglobulin Canx calnexin Rps15 ribosomal protein S15

Forward primer sequence (5' --> 3')

Reverse primer sequence (5' --> 3')

Tissue

AGATCAGGGAGGATGGAACA CCCTGCCATTGTTAAGACC

TCAAAGTGAGGCGATCCATA TGCTGCTGTTCCTGTTTTC

BAT BAT

Annealing temperature (ᵒC) 57 60

CCGAAGTGCAACCCACCATGGG ACTGCTAGCGCGATACATCC TACACCCCGGAACCAGATAC TGCACACATACAGACGGTGCAGC AAAGATCAATCGGACCCTAGACA AGTTAGAGCAGGACAAGCCCAAG TTGTCGGTGACATCCCTTGG ACTCCACTGCTCCAACACCTG ACTTCGAGGAGTTCCTTGCG

CCTTCACCTTGGATCTGAAGGCGGA CATCCAGCCCATGTAGTCCC CAAAGGATTTCACACACGCC GCAGAACCGGAGTCCAGACGT CAGCGAGTAGCGCATAGTCA GTGCAGAGCTGTGCTCCTGA TTGGCCTTGGTCTGGATCAC TGATAGCGTCTGTCCCATAACCTG TGCTACTCCTTGTGGCTGTC

BAT/WAT WAT WAT WAT WAT WAT WAT WAT WAT

60 58 58 60 57 58 62 60 60

CCCCACTGAGACTGATACATACGC GCAGCGACCTATGATTGACAACC CGGAGATGGTGGGTAGCATGG

AGAAACTGGATTTGTAATTAAGCAGGTTC BAT/WAT GCTCCAAACCAATAGCACTGAAAGG BAT ACGGGTTTGTAGGTGATGGAGAAC WAT

60 58 60

2

Supplementary table 2. Microarray pathway analysis of gene profiles of eWAT of 22ºC housed mice compared to 29ºC housed mice. Gene symbol Most changed genes Cck Tph2

Gene name

Systematic name

cholecystokinin tryptophan hydroxylase 2

FC

P

NM_031161 NM_173391

-8.00 -4.91

0.015 0.011

deiodinase, iodothyronine, type II PR domain containing 16 cell death-inducing DNA fragmentation factor, alpha subunit-like effector A uncoupling protein 1 (mitochondrial, proton carrier)

NM_010050 NM_027504

-1.51 1.03

0.289 0.985

NM_007702

1.22

0.911

NM_009463

below detection

NM_011569 NM_011653 NM_027828 NM_011654 NM_009447 NM_019645 NM_001081390 NM_009423

Capza1

tektin 1 tubulin, alpha 1A family with sequence similarity 110, member C tubulin, alpha 1B tubulin, alpha 4A plakophilin 1 palladin, cytoskeletal associated protein TNF receptor associated factor 4 capping protein (actin filament) muscle Z-line, alpha 1

Map1lc3b Coro2b

Browning Dio2 Prdm16 Cidea Ucp1

Cytoskeleton Tekt1 Tuba1a Fam110c Tuba1b Tuba4a Pkp1 Palld Traf4

Cell migration/ growth Igfals Insig1 Cspg4 Cdkn2b Setd8 Hdac9 E2f2 Parm1 Igf1 Plxnd1 Dph6 Zfp467 Klhl21 Larp6 Fgfr1 Pdgfra Srgap1 Klf9 Ppfibp1 Antxr1 Twist2 Thbs2 Inflammation Hp Slc25a1 Tm7sf2 Rab7l1 Twist2 Vnn1

-2.78 -2.27 -1.84 -1.54 -1.53 -1.51 -1.37 -1.33

0.018 0.012 0.013 0.012 0.003 0.036 0.014 0.023

NM_009797

1.11

0.034

microtubule-associated protein 1 light chain 3 beta NM_026160 coronin, actin binding protein, 2B NM_175484

1.15 1.32

0.037 0.040

NM_008340 NM_153526 NM_139001

-1.94 -1.77 -1.61

0.019 0.018 0.012

NM_007670

-1.52

0.018

SET domain containing (lysine methyltransferase) 8 histone deacetylase 9 E2F transcription factor 2 prostate androgen-regulated mucin-like protein 1 insulin-like growth factor 1 plexin D1 diphthamine biosynthesis 6 zinc finger protein 467 kelch-like 21 La ribonucleoprotein domain family, member 6 fibroblast growth factor receptor 1 platelet derived growth factor receptor, alpha polypeptide SLIT-ROBO Rho GTPase activating protein 1 Kruppel-like factor 9 PTPRF interacting protein, binding protein 1 (liprin beta 1) anthrax toxin receptor 1 twist basic helix-loop-helix transcription factor 2 thrombospondin 2

NM_030241 NM_024124 NM_177733 NM_145562 NM_010512 NM_026376 NM_025675 NM_001085417 NM_001033352 NM_026235 NM_010206

-1.43 -1.42 -1.41 -1.36 -1.28 -1.28 -1.19 1.22 1.22 1.23 1.29

0.024 0.018 0.027 0.040 0.012 0.015 0.024 0.042 0.024 0.008 0.048

NM_011058 NM_001081037 NM_010638

1.33 1.33 1.33

0.026 0.018 0.042

NM_001170433 NM_054041 NM_007855 NM_011581

1.36 1.37 1.42 1.60

0.029 0.008 0.002 0.040

haptoglobin solute carrier family 25 (mitochondrial carrier, citrate transporter), member 1 transmembrane 7 superfamily member 2 RAB29, member RAS oncogene family twist basic helix-loop-helix transcription factor 2 vanin 1

NM_017370

-1.90

0.027

NM_153150 NM_028454 NM_144875 NM_007855 NM_011704

-1.54 -1.38 -1.30 1.42 2.03

0.045 0.013 0.037 0.002 0.019

insulin-like growth factor binding protein, acid labile subunit insulin induced gene 1 chondroitin sulfate proteoglycan 4 cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

3

Gene symbol Inflammation: M1 RNA markers S100a8 Saa3 Saa1 Ccl8 Cx3cr1 Ccl19 Ccr2 Ccl2 Il1b Il6 Inflammation: M2 RNA markers Mrc1 Clec10a Cd163 Arg1 Retnla Glucose metabolism Gys2 Grb14 Glyctk Alx3 Acly Nudt14 Slc2a1 Pdk4

Gene name

Systematic name

S100 calcium binding protein A8 (calgranulin A) serum amyloid A 3 serum amyloid A 1 chemokine (C-C motif) ligand 8 chemokine (C-X3-C motif) receptor 1 chemokine (C-C motif) ligand 19 chemokine (C-C motif) receptor 2 chemokine (C-C motif) ligand 2 interleukin 1 beta interleukin 6

FC

P

NM_013650 NM_011315 NM_009117 NM_021443 NM_009987 NM_011888 NM_009915 NM_011333 NM_008361 NM_031168

-4.31 -3.49 -2.91 -2.86 -2.40 -2.06 -1.55 -1.48 -1.38 -1.05

0.002 0.010 0.012 0.002 0.550 0.008 0.111 0.366 0.304 0.968

mannose receptor, C type 1 C-type lectin domain family 10, member A CD163 antigen arginase, liver resistin like alpha

NM_008625 NM_010796 NM_001170395 NM_007482 NM_020509

-1.17 -1.12 -1.06 -1.01 1.12

0.844 0.906 0.980 0.997 0.988

glycogen synthase 2 growth factor receptor bound protein 14 glycerate kinase aristaless-like homeobox 3 ATP citrate lyase nudix (nucleoside diphosphate linked moiety X)type motif 14 solute carrier family 2 (facilitated glucose transporter), member 1 pyruvate dehydrogenase kinase, isoenzyme 4

NM_145572 NM_016719 NM_174846 NM_007441 NM_001199296

-2.22 -1.39 -1.38 -1.35 -1.22

0.002 0.032 0.036 0.015 0.433

NM_025399

-1.22

0.024

NM_011400 NM_013743

-1.00 2.14

0.999 0.015

NM_001080940 NM_009128 NM_012044 NM_007988

-2.33 -2.13 -2.05 -1.86

0.009 0.038 0.008 0.119

NM_153150 NM_025558 NM_013495 NM_026405 NM_001199296 NM_010046 NM_009949 NM_007408 NM_025836 NM_001110149

-1.54 -1.53 -1.44 -1.35 -1.22 -1.05 -1.02 -1.01 -1.01 1.01

0.045 0.028 0.914 0.045 0.433 0.983 0.993 0.997 0.996 0.993

NM_011396 NM_015729 NM_007382 NM_001113471 NM_010719 NM_001159556 NM_020568 NM_176840 NM_024406 NM_001163689 NM_025874

1.03 1.03 1.08 1.08 1.17 1.19 1.22 1.25 1.27 1.34 1.36

0.978 0.988 0.930 0.979 0.925 0.627 0.930 0.024 0.813 0.385 0.485

NM_011977

1.39

0.028

Fatty acid metabolism/beta oxidation Gm6484 angiopoietin-like 8 Scd2 stearoyl-Coenzyme A desaturase 2 Pla2g2e phospholipase A2, group IIE Fasn fatty acid synthase Slc25a1 solute carrier family 25 (mitochondrial carrier, citrate transporter), member 1 Cyb5b cytochrome b5 type B Cpt1a carnitine palmitoyltransferase 1a, liver Rab32 RAB32, member RAS oncogene family Acly ATP citrate lyase Dgat1 diacylglycerol O-acyltransferase 1 Cpt2 carnitine palmitoyltransferase 2 Plin2 perilipin 2 Plin3 perilipin 3 Mgat1 mannoside acetylglucosaminyltransferase 1 Slc22a5 solute carrier family 22 (organic cation transporter), member 5 Acox1 acyl-Coenzyme A oxidase 1, palmitoyl Acadm acyl-Coenzyme A dehydrogenase, medium chain Plin1 perilipin 1 Lipe lipase, hormone sensitive Cd36 CD36 antigen Plin4 perilipin 4 Osbpl11 oxysterol binding protein-like 11 Fabp4 fatty acid binding protein 4, adipocyte Pnpla2 patatin-like phospholipase domain containing 2 Plin5 perilipin 5 solute carrier family 27 (fatty acid transporter), Slc27a1 member 1

Expression of the 2 strongest regulated genes, and key genes for browning, tissue remodelling, inflammation, and metabolism. Genes are ranked based on FC per subgroup. Underlined genes are key markers for the assigned processes. FC = fold change, P = Benjamini-Hochberg false discovery rateadjusted p-value of Student’s t-test.