Fig 2: Identification of TN as a fat-enriched secreted molecule in mice.(A to D) TN mRNA (A and B) or protein (C and D) expression in various human (A and C) or male mouse (B and D) metabolic tissues were determined by qPCR or Western blot, respectively. TN mRNA expression during 3T3-L1 adipocyte (E) and BFC (F) differentiation. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with day 0 during adipocyte differentiation. Values are expressed as means ± SEM. (G and H) Western blot showing the TN protein levels in culture medium (CM) or cell lysates (Cell) during 3T3-L1 adipocyte (G) and BFC (H) differentiation. Adiponectin or UCP1 is shown as a differentiation marker of 3T3-L1 adipocytes or BFC, respectively. (I) TN protein levels in CM and cell lysates of mature 3T3-L1 adipocytes treated with a range of glucose concentrations for 48 hours. Data in (A) to (I) are representative of three independent experiments with similar results. Data are expressed as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3: UCP1, PGC1 alpha and HSL expression in histological sections of hRAN and hRAT. UCP1, PGC1 alpha and HSL expression was evaluated by immunohistochemistry in serial cuts of hRAN and hRAT. DAB staining quantification in the three tissue types was performed with Image J software (NIH). Histograms show mean ± SEM of five independent experiments. (a.u.: arbitrary units). *p < 0.01 hRAN versus hRAT. Representative photographs of hRAN- and hRAT-staining. Magnification: 10× and 40×. Arbitrary units (a.u) represent pixel quantification.

Fig 4: Fat-specific Hlx knockdown and overexpression through viral injection remodel WAT in vivo. a iWAT of wild-type mice was injected with Hlx knockdown lentiviruses. Protein and mRNA expression were analyzed (n = 3 mice per group). b H&E staining of iWAT injected with Hlx knockdown lentiviruses. Shown are representative images of three mice per group. Scale bar, 200 µm. c, d Ucp1 staining (c) and MitoTracker staining (d) of iWAT injected with Hlx knockdown lentiviruses. Shown are representative images of three mice per group. Blue, DAPI. Scale bar, 200 µm. e iWAT of wild-type mice was injected with Hlx overexpression adenoviruses. Protein and mRNA expression were analyzed (n = 4 mice per group). f H&E staining of iWAT injected with Hlx overexpression adenoviruses. Shown are representative images of three mice per group. Scale bar, 200 µm. g, h Ucp1 staining (g) and MitoTracker staining (h) of iWAT injected with Hlx overexpression adenoviruses. Shown are representative images of three mice per group. Blue, DAPI. Scale bar, 200 µm. All injections were repeated with independent cohorts of mice. Tom20 staining was also performed in (d) and (h) and similar results were obtained. All error bars represent s.e.m. Two-tailed unpaired Student’s t-test was performed. **p < 0.01; ***p < 0.001

Fig 5: UCP1: mCherry H1 hES cell line design and integration confirmation.(A) Gene targeting strategy and reporter design.(B) Surveyor nuclease assay of three guide RNAs targeting the UCP1 stop codon. Red arrows indicate expected bands.(C) Identification of targeted ES clones after puromycin selection. PCR primers external and internal to the reporter respectively were used to detect integration. 2 clones were identified, with clone 1 selected for further study.(D) DNA gel demonstrating integration of reporter at UCP1 stop codon in clone 1. External and internal primers were designed for 5’ and 3’ ends of reporter. Left gel indicates integration of 5’ side, with the right gel indicating integration on the 3’ end. Non-targeted HI hES cells were used as a negative control.(E) Cre mediated excision of loxp-pgk-puro cassette from clone 1. 8 sub-clones were identified indicating removal of selection cassette. Sub-clone 5 was selected for further study.(F) Sub-clone 5 is homozygous for the reporter on both alleles. (Left panel) Gel demonstrates lack of the wild type band in sub-clone 5 vs wild type H1 hES cells using primers flanking the UCP1 stop codon. (Right panel) Copy number assay confirms homozygous status of sub-clone 5 vs wild type H1 hES cells.