At the endogenous expression level of USP7 in hASCs was gradually elevated within 15 days after osteogenic induction. A similar pace was observed for the osteogenic marker RUNX2, the master transcription factor of osteogenic differentiation (Fig. 1c). Moreover, the dynamic pattern of protein abundance of these genes was also TariquidarMedChemExpress Tariquidar consistent, as indicated by Western blotting analysis (Fig. 1d), pointing to a role of USP7 in osteogenic differentiation.Knockdown of USP7 inhibits osteogenic differentiation of hASCs in vitroThe cell viability was evaluated with a Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan). Cells were seeded at 5 ?103 cells per well in 48-well plates and cultured in PM or PubMed ID: OM with indicated treatment. At each time point, the supernatant of each group was removed, and cells were incubated with DMEM medium containing CCK-8 for 2 h at 37 . Optical density (OD) was measured at 450 nm using a microplate reader (ELX808, BioTek). Cell apoptosis was examined with Annexin V-FITC Apoptosis Detection Kit (Dojindo, Kumamoto, Japan) according to the manufacture’s protocol. Analysis was performed with a FACScan flow cytometer (Beckman Coulter, CA, USA) using the EXPO32 ADC software.Statistical analysisStatistical results were analyzed by SPSS 20.0 (IBM, Armonk, NY, USA) software. Data from triplicate in vitro experiments were presented as mean ?standardTo validate the potential role of USP7 in osteogenic differentiation of hASCs, we then generated hASCs stably expressing USP7 shRNAs. In this regard, two shRNAs targeting distinct regions of USP7 were designed to limit possible off-target effects. After osteogenic stimulation for 7 days, ALP activity in USP7-deficient cells was significantly decreased compared to control cells (Fig. 2b). Meanwhile, extracellular matrix mineralization, manifested by Alizarin red S staining and quantification on day 14, was also markedly reduced in USP7-deficient cells (Fig. 2b). The knockdown efficiency was analyzed by qRT-PCR and Western blotting, both of which showed that 70 of endogenous USP7 was eliminated compared with the control treatment (Fig. 2a). These results indicated that USP7 is essential for osteogenic differentiation of hASCs. Next, we investigated whether impairment of osteogenic differentiation resulting from USP7 deficiency isTang et al. Stem Cell Research Therapy (2017) 8:Page 5 ofabcdFig. 1 Protein deubiquitinase USP7 is potentially associated with hASC osteogenesis. a Schematic diagram of OCpro-Luc-hASC construction. b OCpro-Luc-hASCs were transfected with siRNA against USP7, with the scrambled siRNA as negative control and siRNA against RUNX2 as positive control. Left panel: Luciferase reporter assay with cellular extracts from OCpro-Luc-hASCs cultured in proliferation or osteogenic media for 7 days. Middle and right panel: Validation of USP7 and RUNX2 knockdown effect by Western blotting, respectively. c Relative mRNA expression of USP7 and RUNX2 measured by qRT-PCR during osteogenic differentiation of hASCs (normalized by GAPDH; relative to day 0 groups). d Western blotting of protein expression of USP7, RUNX2, and the internal control GAPDH during osteogenic differentiation of hASCs. Results are presented as the mean ?SD, n = 3. *P < 0.05, **P < 0.01. hASC human adipose-derived stem cell, Luc luciferase, NC negative control, OC osteocalcin, OM osteogenic media, PM proliferation media, RUNX2 runt-related transcription factor 2, USP7 ubiquitin specific proteaseassociated with a.