1. Volume 26, Issue 12, Pages 3221-3230.e3 (March 2019)
Prolyl Isomerase Pin1 Suppresses Thermogenic Programs in Adipocytes by Promoting Degradation of Transcriptional Co-activator PRDM16 
Yusuke Nakatsu, Yasuka Matsunaga, Takeshi Yamamotoya, Koji Ueda, Masa-ki Inoue, Yu Mizuno, Mikako Nakanishi, Tomomi Sano, Yosuke Yamawaki, Akifumi Kushiyama, Hideyuki Sakoda, Midori Fujishiro, Akihide Ryo, Hiraku Ono, Tohru Minamino, Shin-Ichiro Takahashi, Haruya Ohno, Masayasu Yoneda, Kei Takahashi, Hisamitsu Ishihara, Hideki Katagiri, Fusanori Nishimura, Takashi Kanematsu, Tetsuya Yamada, Tomoichiro Asano 
Cell Reports 
Volume 26, Issue 12, Pages e3 (March 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 3221-3230.e3DOI: (10.1016/j.celrep.2019.02.066)
Copyright © 2019 The Authors Terms and Conditions

3. Figure 1
Deficiency of Pin1 in Adipocytes Protects against Diet-Induced Obesity
(A–D) BAT and scWAT were both extracted from 16-week-old male controls, ob/ob mice (A and B), and mice fed an HFD for 8 weeks (C and D). Then, Pin1 and actin protein levels were determined by western blotting (n = 4).
(E) Changes in body weight in Pin1 flox and AdPin1 KO were measured after HFD feeding (n = 5–6).
(F) Each tissue was extirpated from mice 8 weeks after the start of HFD feeding and weights were measured.
(G) Liver triglyceride levels were measured (n = 5–6).
(H) H&E staining.
(I and J) Glucose tolerance (I) and insulin tolerance (J) tests were performed using mice that had been fed the HFD for 6 or 7 weeks (n = 5–6).
Data are expressed as means + SEMs. ∗p < 0.05, ∗∗p < 0.01.
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4. Figure 2
Pin1 Ablation in BAT and scWAT Promotes Thermogenesis in Response to Cold Exposure
(A and B) BAT (A) and scWAT (B) were both prepared from Pin1 flox or AdPin1 KO mice 6 h after cold shock or RT conditions. Relative mRNA levels of thermogenic genes were investigated (n = 6–7).
(C and D) UCP-1 and actin protein levels were examined by western blotting in BAT (C) or scWAT (D) (n = 7).
(E and F) Representative images are shown of H&E and UCP staining of BAT (E) or scWAT (F) sections.
(G) Averages of O2 consumption during the light and dark periods (n = 5) are shown.
Data are expressed as means + SEMs. ∗p < 0.05, ∗∗p < 0.01.
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5. Figure 3 Pin1 Associates with PRDM16 and Promotes Its Degradation
(A) The molecular mechanism underlying UCP-1 induction is illustrated.
(B) Both FLAG-PRDM16 and S-tag Pin1 were overexpressed in 293T cells. The cell lysates were then immunoprecipitated with FLAG beads.
(C) Cell lysates were prepared from mouse scWAT and then immunoprecipitated with Pin1 antibody.
(D) FLAG-PRDM16 was overexpressed in 293T cells and cell lysates were prepared. The cell lysates were incubated with glutathione S-transferase (GST) proteins.
(E) FLAG-tagged PRDM16 deletion mutants were overexpressed with S-tag Pin1 in 293T cells. Next, immunoprecipitation was performed using FLAG beads.
(F) FLAG-PRDM16 was overexpressed with wild S-tag Pin1 or K63A Pin1 mutant in 293T cells. K63A, PPIase inactive form.
(G) 293T cells were treated with negative siRNA or Pin1 siRNA. Then, FLAG-PRDM16 was overexpressed.
(H) 293T cells overexpressing PRDM16 were treated with or without 10 μM MG-132 for 8 h.
(I) 293T cells were treated with siRNA. Then, plasmid transfection was performed, and the cells were treated with or without MG-132.
(J) 293T cells were transfected with PPARγ, PRDM16, PPAR-responsive elements-luciferase (PPRE-Luc), and TK-rluc. After 48 h, luciferase assays were performed (n = 4).
Data are expressed as means + SEMs. ∗∗p < 0.01.
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6. Figure 4
Pin1 Deletion in Primary Adipocytes Enhances Thermogenic Programs in Response to Treatment with CL316243
(A) Stromal vascular fractions (SVFs) were prepared from Pin1 flox or AdPin1 KO mice, and then differentiated into mature adipocytes. Oil red O staining was performed to identify lipid droplets.
(B and C) Brown (B) and subcutaneous white (C) adipocytes were exposed to 1 μM CL for 6 h. Then, RNA was extracted (n = 4).
(D) Mature adipocytes were exposed to CL for 6 h. Then, UCP-1 protein levels were examined by western blotting.
(E) Proteins were extracted from mature adipocytes. Then, PRDM16 expression levels were examined.
(F) SVFs were treated with siRNA and then differentiated into mature adipocytes. On day 4, adipocytes were stimulated with CL for 6 h. N, negative siRNA; PR, PPRDM16 siRNA.
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