Supplementary Figure 1. Nur77 and leptin-controlled obesity. (A) (B) (C)

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1 Supplementary Figure 1. Nur77 and leptin-controlled obesity. (A) Effect of leptin on body weight and food intake between WT and KO mice at the age of 12 weeks (n=7). Mice were i.c.v. injected with saline for 2 days and then with leptin (1 μg) for 6 days. (B) Comparison of WAT and liver weight in WT and KO mice with and without leptin treatment (n=6). Mice were i.c.v. injected with leptin (1 μg) for 6 days. Two hours after final leptin injection, mice were euthanized and intra-abdominal WAT (left) and liver (right) were weighted and corrected by body weight. (C) Comparison of related gene expression levels in EWAT (top) and liver (bottom) between WT and KO mice with and without leptin i.c.v. injection (n=6). RNA were prepared from EWAT and liver and processed for real-time PCR. All data are presented as means with SEM of two or three independent results. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

2 Supplementary Figure 2. Attenuated leptin sensitivity in Nur77 KD mice. (A) Top, a typical image on the high infection efficiency in ARC of hypothalamus after adenovirus injection. 3V, the third ventricle; ARC, arcuate nucleus. Bottom, western blot shows the efficiency of Nur77 knockdown in different tissues (n=6). Twelve-week-old mice were bilaterally injected with adenovirus-packed scramble-shrna (Ctrl) or Nur77-shRNA (KD) into the mediobasal hypothalamus. One week later, hypothalami (H), muscle (M), liver (L) and EWAT (W) were extracted. Supplementary Figure 3. Modulation of chronic leptin resistance by Nur77 in HFD mice. (A) After leptin injection, livers of HFD-fed WT and Nur77 KO mice were weighted and corrected by body weight (left, n=6), and then processed for Oil red staining (right, Scale bar, 50 μm). Data are presented as the means with SEM of three independent results. ns: no significance.

3 Supplementary Figure 4. Effect of Nur77 on leptin-induced STAT3 acetylation in cell system. (A) Interactions between Nur77 and STAT3. Left, HEK293T cells were transfected with Flag-STAT3 and Myc-Nur77, Co-IP assay was performed to determine their interactions. Middle, 12-week-old mice were fasted for 18 hours and then i.p. injected with saline or leptin (1 μg/g, 30 min, n=6). Hypothalami were extracted for Co-IP assay. Right, GT1-7 cells were serum starved overnight and then subjected to 1 mm glucose DMEM for 6 hours. After treated with leptin (0.5 g/ml, 30 min), cells were harvested and subjected to Co-IP assay. (B) Nur77 was overexpressed by transfection of HA-Nur77 (top), or knocked down by lentivirus-based RNA interference (bottom) in AtT-20 cells (left) or GT1-7 cells (right). Cells were treated with leptin (0.5 g/ml, 30 min) and acetylation of STAT3 was then detected by IP/western blot. Scramble-shRNA or empty vector was used as a negative control. (C) Effect of Nur77 on OB-Rb expression. WT and Nur77 KO mice were fasted for 18 hours and then injected with leptin (1 g/g, 30 min) and OB-Rb expression was detected by western blot. (D) Effect of acetylases (p300 and SRC1) and deacetylases (SirT1 and HDAC1) on Nur77-induced STAT3 activity. Various plasmids were introduced into AtT-20 cells (left) and GT1-7 cells (right) as indicated, and then treated with leptin (0.5 g/ml) for 6 hours. The luciferase assays were performed. (E) Knockdown (left) or overexpression (right) of Nur77 impairs p300 or HDAC1 effect on STAT3 activity in AtT-20 (top) and GT1-7 cells (bottom). Nur77 was knocked down by the lentivirus-based RNA interference. Various plasmids were introduced into AtT-20 cells (left) and GT1-7 cells (right) as indicated, and then treated with leptin (0.5 g/ml) for 6 hours. The luciferase assays were performed. Data are presented as the means with SEM of three independent results. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

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6 Supplementary Figure 5. Effect of Nur77 on downstream genes of leptin signaling. (A) Comparison of gene expression levels of NPY and AgRp between WT and Nur77 KO mice of 12 weeks (n=6). Mice were fasted for 18 hours and then treated with leptin (1 g/g, i.p.) for 2 hours. Hypothalami were extracted and processed for real-time PCR. The data are presented as the means with SEM of three independent results. * p < 0.05, ns: no significance.

7 Supplementary Figure 6. Corresponding parameters in age-induced obese WT and Nur77 KO mice. (A) Cold exposure tests were performed in WT and KO mice at the age of 12 and 40 weeks. Mice were settled in cages without bedding and housed into 4 C for 0, 30, 60, 120 and 180 min (n=6). The body temperature was recorded respectively. (B) Comparison of the serum levels of leptin between WT and KO mice at the age of 12 and 40 weeks (n=6). (C) Comparison of Pomc gene expression between 12- or 40-week-old WT and Nur77 KO mice. Hypothalami were isolated and processed for real-time PCR and ISH assay (n=6). 3V, the third ventricle; ARC, arcuate nucleus. (D) Left, haematoxylin and eosin staining indicated the morphology of BAT in WT and Nur77 KO mice at the age of 40 weeks under a microscope (scale bar, 10 μm). Right, comparison of related genes in BAT of 40-week-old WT and Nur77 KO mice (n=6). All data are presented as means with SEM of two or three independent results. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

8 Supplementary Table 1. Primers used for mutants of STAT3 and pgl3-pomc generation (the mutated sites were marked with a line) Mutants Forward primers (5-3 ) Reverse primers (5-3 ) STAT3 K49R ATGCAGCCAGCAGAGAGTCACATGCCACGTTGGTG TGGCATGTGACTCTCTGCTGGCTGCATATGCCCAGTC STAT3 K87R CCTTCGAAGAATCAGGCAGTTTCTGCAGAGCAGGTATCTTG CTGCAGAAACTGCCTGATTCTTCGAAGGTTGTGCTGATAG STAT3 K685R GAGGCATTTGGAAGGTACTGTAGGCCCGAGAGCCAG CGGGCCTACAGTACCTTCCAAATGCCTCCTCCTTGG STAT3 K49Q ATATGCAGCCAGCCAAGAGTCACATGCCACGTTGGTG TGGCATGTGACTCTTGGCTGGCTGCATATGCCCAGT STAT3 K87Q AACCTTCGAAGAATCCAGCAGTTTCTGCAGAGCAGGTATCT CTGCAGAAACTGCTGGATTCTTCGAAGGTTGTGCTGATAGA STAT3 K685Q GGAGGCATTTGGACAGTACTGTAGGCCCGAGAGCCAG GGGCCTACAGTACTGTCCAAATGCCTCCTCCTTGGG Pomc NurRE mutant AACAAATCCCCTTCCTCATTAGTGATAGCGACCTCCGGGTG CCAGGAAGGC GTCCATCTGCCTTCCTGGCACCCGGAGGTCGCTATCACTAA TGAGGAA Pomc NBRE mutant GCAGAAGCGCTGCCAGGAATTCCACGTCCAAGGC GTGGGTGAGCCTTGGACGTGGAATTCCTGGCAGCG Pomc SPRE mutant CCTCCCGAGGCAAGAAGCCCCCCTTCGCTGCAG GAAGGGGGGCTTCTTGCCTCGGGAGGGGGGC Pomc STRE mutant GTGATATTTACCTCCAAATGCCAGGGGGGCAGATGGACG TACCTATGTGCGTCCATCTGCCCCCCTGGCATTTGG Supplementary Table 2. Oligonucleotides sequences for shrna and primers used for SeqChIP Oligonucleotides sequences for shrna (5-3 ): Nur77-shRNA STAT3-shRNA GGGCATGGTGAAGGAAGTT GGGTGAAATTGACCAGCAA Scramble-shRNA GCGCGCTTTGTAGGATTCG Primers used for SeqChIP (5-3 ): Distal Pomc promoter CTCTGTTGTCTCCCTTCTCA CCTATGTGCGTCCATCTGCC Proximal Pomc promoter TGTGCTAACGCCAGCCTCCG TTTGGTCCCTGTCGCTCTTC

9 Supplementary Table 3. Primers used for quantitative real-time PCR Genes Forward primer(5'-3') Reverse primer(5'-3') Nur77 AACCCAAGCAGCCTCCAGAT CTGCCCACTTTCGGATAACG STAT3 CAGTCGGGCCTCAGCCC AGGACATTGGACTCTTGCAG Pomc AGGCCTGACACGTGGAAGAT AGGCACCAGCTCCACACAT NPY TAACAAGCGAATGGGGCTGT ATCTGGCCATGTCCTCTGCT AgRp TGCTACTGCCGCTTCTTCAA CTTTGCCCAAACAACATCCA PGC1α CTGCTGATGCCTGTTGTC AGTGTCTTCGGGATTGTC SREBP1c CCTTAACGTGGGCCTAGTC TGTCCAGTTCGCACATCTC FASN CGGCTTCGCCAACTCTACCA CCCATCGCTTCCAGGACAAT ACC CAGTTCAGTCTCGGTGCT TCTTGGACAAATGGGTAGT SCD1 CAAGAACATTCAATCCCGGGAG CAGGGCAGCCATGCAGTC CPT1 CCTTGCTTGATTCCTACTC ATGTTGCCTTTTGTTTTCT HSL GACCCAGAGTTGCGTGGGGC GCTGAGCAGGCGGCTTACCC UCP1 ACGTCCCCTGCCATTTACTG GGTACGCTTGGGTACTGTCC UCP2 ACTTCACTTCTGCCTTCGGG AGGAAGGCATGAACCCCTTG UCP3 CCTACGACATCATCAAGGAGAAGTT TCCAAAGGCAGAGACAAAGTGA β-actin GATGGCCACTGCCGCATCCTC GGTCTTTACGGATGTCAACGTCAC Supplementary Table 4. Antibody used in Co-IP and western blot analysis Antibody Cat. no. Company Anti-STAT3 Antibody 4904S Cell signaling Anti-Phospho-STAT3 (Tyr705) antibody 9145 Cell signaling Anti-Nur77 antibody for human Nur Cell Signaling Anti-Nur77 antibody for mouse Nur Epitomics Anti-Acetylated-Lysine antibody Millipore Anti-OB-Rb antibody AF497 R&D Anti-Tubulin antibody T-4026 Sigma Anti-Flag antibody F-3165 Sigma Anti-HA antibody H-9658 Sigma