Sirt1 exerts anti-inflammatory effects and promotes steroidogenesis in Leydig cells  Ling Wu, M.Sc., Aijun Zhang, Ph.D., Yijuan Sun, Ph.D., Xiaobin Zhu, M.Sc., Weimin Fan, M.Sc., Xiaowei Lu, M.Sc., Qingqing Yang, M.Sc., Yun Feng, M.D., Ph.D.  Fertility and Sterility  Volume 98, Issue 1, Pages 194-199 (July 2012) DOI: 10.1016/j.fertnstert.2012.04.008 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Down-regulation of Sirt1 expression by proinflammatory cytokines. (A, B) Western blot analysis was performed using the protein extracts from C57BL/6 mice under normal diet (A) and cell lines (B). SM = skeletal muscle; WAT = white adipose tissue. (C, D) mRNA and protein levels of Sirt1 were determined by real-time PCR (C) and Western blot (D) analysis. TM3 cells were treated with control (PBS) or TNFα (10 ng/mL) for 24 hours. (E) Real-time PCR analysis of Sirt1 expression in TM3 cells treated with control (PBS), IL-6 (20 ng/mL), or IL-1β (2 ng/mL). (F) C57BL/6 mice fed a normal diet were treated with control (saline) or LPS (5 mg/kg) for 8 hours. Sirt1 mRNA levels in the testis were analyzed by real-time PCR (n = 5). (G) Sirt1 mRNA levels in the testis of C57BL/6 mice fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks (n = 6). *P<.05, **P<.01. Fertility and Sterility 2012 98, 194-199DOI: (10.1016/j.fertnstert.2012.04.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of Sirt1 agonist or overexpression on cytokine-induced cytotoxicity. (A, B) The cell viability (A) and cell proliferative potential (B) were determined in TM3 cells treated with control (DMSO) or Res (25 μM). **P<.01 vs. DMSO + PBS; &P<.01 vs. DMSO + TNFα or IL-1β. (C) Western blot analysis of Sirt1 expression in TM3 cell transfected with empty vector (EV) or Sirt1 expression plasmid. (D, E) The cell viability (D) and cell proliferative potential (E) were determined in TM3 cells transfected with empty vector (EV) or Sirt1 expression plasmid (**P<.01 vs. EV + PBS; &P<.01 vs. EV + TNFα or IL-1β). Fertility and Sterility 2012 98, 194-199DOI: (10.1016/j.fertnstert.2012.04.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Sirt1 exerts its anti-inflammatory roles in TM3 cells. (A, B) Real-time PCR and Western blot analysis of TM3 cells transfected with nonspecific control siRNA (Ctrl) or two Sirt1 siRNA oligos. (C) mRNA expression levels of the indicated genes in TM3 cells after transfection of control siRNA or Sirt1 siRNA. (D) Effect of Sirt1 overexpression on TNFα-induced IL-6, iNOS, and Cox2 expression in TM3 cells. iNOS = inducible nitric oxide synthase; Cox2 = cyclooxygenase-2. *P<.05, **P<.01, ***P<.001. Fertility and Sterility 2012 98, 194-199DOI: (10.1016/j.fertnstert.2012.04.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Sirt1 acts as a coactivator of steroidogenic factor 1. (A) Sirt1 interacted with SF-1. SF-1 was pulled down by Sirt1 by immunoprecipitation using anti-Flag M2 beads in HEK293T cells cotransfected with Flag-Sirt1 and GFP-SF-1. SF-1: steroidogenic factor 1. (B, C) The luciferase reporter assay for SF-1 transcription activity. SF-1 transcription activity was enhanced by Sirt1 overexpression in StAR promoter (B) and Cyp11B1 promoter (C). StAR = steroidogenic acute regulatory protein; CYP11B1 = cytochrome P450, family 11, subfamily B, polypeptide 1. (D, E) mRNA levels of the indicated steroidogenic enzymes in TM3 cells transfected with Sirt1 siRNA oligos (D) or Sirt1 expression plasmid (E). P450c17 = steroid 17-alpha-hydroxylase/17, 20 lyase; 3β-HSD = hydroxy-delta-5-steroid dehydrogenase. *P<.05, **P<.01. Fertility and Sterility 2012 98, 194-199DOI: (10.1016/j.fertnstert.2012.04.008) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions