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. 2016 Mar 18;23(8):1296–1311. doi: 10.1038/cdd.2016.6

Figure 1.

Figure 1

Figure 1

NEDD8 induction is essential for adipocyte differentiation. (a) NEDD8 and adipogenic transcription factors are induced in 3T3-L1 cells under differentiation. 3T3-L1 cells were stimulated with DMI for the indicated time, and protein expression was evaluated by western blotting. (b) Adipocyte differentiation is retarded by blocking neddylation. 3T3-L1 cells were transfected transiently with si-Control, si-NEDD8 or si-APPBP1 (10, 20 or 50 nM) and then stimulated with DMI for 8 days. Western blotting (upper panel) and Oil Red O staining (lower panel) were performed. (c) PPARγ and C/EBPs are downregulated by NEDD8 knockdown. 3T3-L1 cells were transfected with 50 nM of si-control or si-NEDD8, stabilized for 2 days, and then stimulated by DMI. C/EBPs and PPARγ protein levels were determined by immunoblot analysis in cell lysates and β-tubulin was used as a loading control. (d) NEDD8 is required for the expressions of adipogenic genes. 3T3-L1 cells were transfected with si-control (Si-Con) or si-NEDD8 (Si-N8) and then stimulated with DMI for the indicated lengths of time. Total RNA was purified from the cells, and RT-qPCR was performed to measure the expression levels of Pparg, Cebpa, Cebpb, Cebpd, Fasn, Fabp4, Cd36, adiponectin, resistin, and Nedd8. Expression levels are shown relative to those of 18S RNA (mean+S.D.). Data are presented as the means+S.D. (n=3); *P<0.05. (e and f) NEDD8 promotes PPARγ binding to the promoters of its target genes. Stable 3T3-L1 cell lines expressing non-targeting shRNA (pLKO.1-sh-Con), NEDD8-targeting shRNA (pLKO.1-sh-N8), pLVX-IRES, and pLVX-IRES-His-N8 vectors were stimulated with DMI, and chromatin was cross-linked and immunoprecipitated using an anti-PPARγ antibody. The precipitated Cebpa (f) and Fabp4 promoters (e) were amplified and quantified by qPCR. The results (mean+S.D., n=3) are expressed as percentages of the input level. PPRE, PPARγ response element; NS, non-specific element. (g) PPARγ protein is expressed depending on NEDD8 levels. Cell lysates stimulated with DMI in the stable 3T3-L1 cell lines expressing non-targeting shRNA (pLKO.1-sh-Con), NEDD8-targeting shRNA (pLKO.1-sh-N8), pLVX-IRES, and pLVX-IRES-His-N8 vectors were analyzed by immunoblot analysis. β-Tubulin was used as a loading control. The intensities of PPARγ protein bands were quantified using Image J. Data are presented as the means+S.D. (n=3); *P<0.05