Supplementary Data

SUPPLEMENTARY DATA

Supplementary Table 1. Primer sequences for qRT-PCR

Gene Primer Sequences Forward 5'-CCA CCA GCG AGG ACT TCA C-3' PRDM16 Reverse 5'-GGA GGA CTC TCG TAG CTC GAA -3' Forward 5'-GTGAACCCGACAACTTCCGAA -3' UCP1 Reverse 5'-TGAAACTCCGGCTGAGAAGAT-3' 5'-TAT GGA GTG ACA TAG AGT GTG CT- Forward PGC1α 3' Reverse 5'-CCA CTT CAA TCC ACC CAG AAA G -3' Forward 5'-AATTATGCCTCGGAGAAGACCG-3' Dio2 Reverse 5'-GGCAGTTGCCTAGTGAAAGGT-3' Forward 5'-TCC GCG TTC TCA TGT AGG TCT-3' Elovl3 Reverse 5'-GGA CCT GAT GCA ACC CTA TGA-3' Forward 5'-TGC TCT TCT GTA TCG CCC AGT-3' Cidea Reverse 5'-GCC GTG TTA AGG AAT CTG CTG-3' Forward 5'-TGT GGG GAT CTC AGC CAT AGT -3' Cox8b Reverse 5'-AGT GGG CTA AGA CCC ATC CTG -3' Forward 5'-TCGCTGATGCACTGCCTATG-3' PPARγ Reverse 5'-GAGAGGTCCACAGAGCTGATT-3' Forward 5'-AAGGTGAAGAGCATCATAACCCT-3' AP2 Reverse 5'-TCACGCCTTTCATAACACATTCC-3' Forward 5'-GGAATGTGGAGCGTGCTAAAA-3' mtTFAM Reverse 5'-GCTGGAAAAACACTTCGGAATA-3' Forward 5'-GGAGGCAAGCATAAGACTGG-3' CyCs Reverse 5'-CCATCAGGGTATCCTCTC-3' Forward 5'-CCCGATTGAAGTAAAGGCTGT-3' Nampt Reverse 5'-TGGTAAGCCAGTAGCACTCTG-3' Forward 5'-AGCACGGAGTGACCCAAAC-3' NRF1 Reverse 5'-TGTACGTGGCTACATGGACCT-3' Forward 5'-CCGCAAGGGAAAGATGAAAGAC-3' 16s-rRNA Reverse 5'-TCGTTTGGTTTCGGGGTTTC-3' Hexokinase Forward 5'-GCCAGCCTCTCCTGATTTTAGTGT-3' 2, intron 9 Reverse 5'-GGGAACACAAAAGACCTCTTCTGG-3' Forward 5'-GCAGGAGTACGATGAGTCCG-3' β-actin Reverse 5'-ACGCAGCTCAGTAACAGTCC-3'

Supplementary Table 2. Primary antibodies for western blotting

Antibodies Company Catalog No. Dilution PRDM16 Abcam ab106410 1:1000 ab23841 1:1000 (WB) UCP1 Abcam ab10983 1:100 (IF) PGC1α Santa Cruz sc-13067 1:1000

Dio2 Abcam Ab77481 1:500 PTEN Santa Cruz sc-7974 1:1000 AMPKα Cell Signaling #2793 1:1000 p-AMPKα (Thr172) Cell Signaling #2535 1:1000 β-actin Santa Cruz sc-47778 1:1000

Supplementary Figure 1. BLC attenuates hepatic lipid accumulation and stimulates the expression of thermogenic gene expressions in the liver. A: Total lipid and triglyceride (TG) contents of livers in the mice. B: Representative images of H&E-stained sections and gross morphology of livers (original magnification, ×200). C: Thermogenic gene expression analyzed by qPCR. Data are mean ± SEM (n=10 per group). *p<0.05, **p<0.01 and ***p<0.001 vs. HFD group.

Supplementary Figure 2. BLC does not induce toxicity. A: Appearance of the experimental mice. B: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in the serum of the experimental mice fed BLC for 11 weeks.

Supplementary Figure 3. Expression of brown adipocyte specific genes in the scWAT of HBLC mice to BAT of HFD mice. Thermogenic gene expression analyzed by RT-qPCR. Data are mean ± SEM (n=10 per group). **p<0.01, ***p<0.001 vs. scWAT-HFD group. #p<0.05, ##p<0.01 vs. scWAT- HBLC group.

Supplementary Figure 4. Cold exposure increases fat browning and mitochondrial function. Male C57BL/6 mice (8 weeks old) were either kept at room temperature (RT) or exposed to 4°C for 4 hours per day during 3 consecutive days (CE). A-D: Expression levels of brown adipocyte-specific genes and proteins and mitochondria function-related genes and proteins were analyzed by qPCR and western blotting, respectively, in the scWAT (A and B) and muscle (C and D) of mice that were exposed to cold (CE) or room temperature (RT). Data are expressed as mean ± SEM (*p<0.05 and **p<0.01 vs. RT group).

Supplementary Figure 5. Expression ratio of brown adipocyte specific genes normalized by AP2. SVFs were differentiated into white adipocyte and analyzed the expression of brown adipocyte specific gene after normalization by adipocyte specific marker, AP2. *p<0.05, **p<0.01 and ***p<0.001 vs. control.

Supplementary Figure 6. BLC induces fat browning through activation of AMPK. SVFs were transfected with ctrl-siRNA or AMPK siRNA for 2 days and differentiated into beige adipocytes for 6 days in the presence and absence of BLC (1 μM). The expression of UCP1 was quantified by western blotting.

Supplementary Figure 7. BLC stimulates differentiation into beige adipocytes through mediation of T3. A-D: SVFs were differentiated into beige adipocytes with BLC or without BLC under T3-deleted conditions and compared to adipocytes under T3-enriched conditions. Quantitative RT-PCR analysis of UCP1 (A), PGC1α (B), and Dio2 (C) mRNA expression, as well as western blotting for UCP1 (D), in SVF-differentiated adipocytes. E: The concentration of T3 in the serum and tissues analyzed by ELISA kit (Calbiotech, Spring Valley, CA). F: SVFs were differentiated into adipocytes in the presence or absence of TR antagonist, 1-850 (5μM). The UCP1 mRNA and protein levels was quantified by qRT-PCR and western blotting. Data are expressed as mean ± SEM.