N Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Tang et al. Stem Cell Research Therapy (2017) 8:Page 2 ofhistone demethylase retinoblastoma binding protein 2 (RBP2) and lysine-specific demethylase 1 (LSD1) inhibited osteogenic differentiation of hASCs through repression of osteogenesis-related gene expression via their catalytic activities [9, 10]. Similar to methylation, protein ubiquitination is also a reversible modification and has been implicated in stem cell differentiation and organ PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 development or homeostasis [11]. In humans, a large family of deubiquitinases (DUBs) act to oppose protein ubiquitination through hydrolyzing the ubiquitin linkages, thereby controlling the function or abundance of targeted proteins and influencing physiological or pathological processes [12, 13]. Among these DUBs, ubiquitin specific protease 7 (USP7), also known as herpes virus-associated ubiquitin-specific protease (HAUSP), is one of the most extensively studied DUBs. Accumulating studies have shown that USP7 is able to target different proteins to regulate a series of biological processes including the immune response [14], virus replication and infection [15], mitosis [16], DNA replication [17], and DNA damage repair [18]. Meanwhile, the abnormal expression or functional dysregulation of USP7 is associated with pathological processes such as neurodegenerative diseases [19], inflammation [20], and tumors [21, 22]. Currently, USP7 has been reported to stabilize the repressor element 1-silencing transcription factor (REST) and is involved in the maintenance of neural progenitor cells [23]. However, the roles of USP7 on fate determination or orientated differentiation of MSCs remain to be investigated. In this study, we Sitravatinib web investigated the previously unrecognized biological and functional roles of protein deubiquitinase USP7 in osteogenic differentiation of hASCs, both in vitro and in vivo, and explored whether USP7 regulated osteogenesis through its catalytic activity. We believe our findings will facilitate the development of a drug or small molecule to act on USP7 to modulate osteogenic differentiation of MSCs.osteogenic media (OM) containing standard PM supplemented with 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM -glycerophosphate. Adipogenic differentiation of cells was induced when cells grew to 100 confluence with adipogenic media (AM) containing standard PM supplemented with 10 M insulin, 1 M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 200 M indomethacin. HBX 41,108 was purchased from Tocris (MN, USA).Reporter vector construction and luciferase reporter assayThe genomic DNA harboring promoter regions of osteocalcin (OCpro) and DNA sequences encoding luciferase genes were amplified and cloned into the pLVX-pTREpuro vector. Then, the generated OCpro-Luc-Puro construct together with three assistant vectors were transiently transfected into HEK293T cells followed by viral supernatant collection, filtr.
