Subjects: Biology >> Biophysics submitted time 2016-05-12
Chen, Xiulan
Wei, Shasha
Ji, Yanlong
Guo, Xiaojing
Yang, Fuquan
Chen, Xiulan
Wei, Shasha
Ji, Yanlong
Guo, Xiaojing
Yang, Fuquan
Ji, Yanlong
Abstract： SILAC is based on direct addition of selected stable isotope amino acids into the cell culture medium, allowing superior quantitative analysis of the cellular proteome compared to other labeling methods. The great advantages of SILAC lie in its straight-forward implementation, quantitative accuracy, and reproducibility over chemical labeling or label-free quantification strategies, favoring its adoption for proteomic research. SILAC has been widely applied to characterize the proteomic changes between different biological samples, to investigate dynamic changes of protein PTMs, to distinguish specific interacting proteins in interaction proteomic analysis, and to analyze protein turnover in the proteome-wide scale. The present review summarizes the principles of SILAC technology, its applications in biological research, and the present state of this technology.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics submitted time 2016-05-12
Shu, Qingbo
Cai, Tanxi
Chen, Xiulan
Xue, Peng
Zhu, Nali
Xie, Zhensheng
Wei, Shasha
Niu, Lili
Yang, Fuquan
Shu, Qingbo
Cai, Tanxi
Chen, Xiulan
Xue, Peng
Zhu, Nali
Xie, Zhensheng
Wei, Shasha
Niu, Lili
Yang, Fuquan
Shu, Qingbo
Zhang, Qing
Abstract： One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AT cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AT cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.
Peer Review Status:Awaiting Review
Subjects: Biology >> Biophysics submitted time 2016-05-05
Chen, Xiulan
Wei, Shasha
Yang, Fuquan
Chen, Xiulan
Wei, Shasha
Yang, Fuquan
Xu, Shimeng
Deng, Yaqin
Li, Yiran
Liu, Pingsheng
Xu, Shimeng
Wei, Shasha
Deng, Yaqin
Li, Yiran
Abstract： Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes.
Peer Review Status:Awaiting Review
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