Knockdown of LAP2α inhibits adipogenesis of human adipose-derived stem cells and ameliorates high-fat diet-induced obesity.

Adipogenesis, a pivotal cellular process involving the differentiation of mesenchymal stem cells (MSCs) to mature adipocytes, plays a significant role in various physiological functions. Dysregulation of adipogenesis is implicated in conditions such as obesity. However, the complete molecular understanding of adipogenesis remains elusive. This study aimed to uncover the novel role of lamina-associated polypeptide 2 alpha (LAP2α) in human adipose-derived stem cells (hASCs) adipogenesis and its impact on high-fat diet (HFD)-induced obesity and associated metabolic disturbances. LAP2α expression was assessed during the adipogenic differentiation of hASCs using RT-qPCR and western blotting. The functional role of LAP2α in adipogenesis was explored both in vitro and in vivo through loss- and gain-of-function studies. Moreover, mice with HFD-induced obesity received lentivirus injection to assess the effect of LAP2α knockdown on fat accumulation. Molecular mechanisms underlying LAP2α in adipogenic differentiation were investigated using RT-qPCR, Western blotting, immunofluorescence staining, and Oil Red O staining. LAP2α expression was upregulated during hASCs adipogenic differentiation. LAP2α knockdown hindered adipogenesis, while LAP2α overexpression promoted adipogenic differentiation. Notably, LAP2α deficiency resisted HFD-induced obesity, improved glucose intolerance, mitigated insulin resistance, and prevented fatty liver development. Mechanistically, LAP2α knockdown attenuated signal transducer and activator of transcription 3 (STAT3) activation by reducing the protein level of phosphorylated STAT3. A STAT3 activator (Colivelin) counteracted the negative impact of LAP2α deficiency on hASCs adipogenic differentiation. Taken together, our current study established LAP2α as a crucial regulator of hASCs adipogenic differentiation, unveiling a new therapeutic target for obesity prevention.

[Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

Clinical feasibility evaluation of a digital workflow of prosthetically oriented onlay bone grafting for horizontal alveolar augmentation: a prospective pilot study.

BACKGROUND: Onlay bone grafting is considered highly reliable for reconstructing severe horizontal bone defects. A critical problem is how to achieve precise position of the bone block to control alveolar ridge dimensions. This research aims to establish a digital workflow for prosthetically oriented onlay bone grafting and evaluate its accuracy and efficiency. METHODS: This prospective pilot study investigated eight patients who required implant restoration in the esthetic area with horizontal alveolar bone defects. The workflow includes preoperative virtual planning, design and manufacture of patient-specific templates, bone grafting surgery, and implant insertion. Primary outcomes were graft accuracy, defined by root mean square estimate (RMSE) values between preoperatively designed and actual implanted outer contours of bone blocks. Secondary outcomes were bone graft and implant success rates. Besides, the surgeons used the visual analog scale (VAS) to rate the intuitiveness, ease of understanding, and helpfulness of the workflow. RESULTS: No bone grafts or implants failed in any of the eight patients, resulting in a 100% success rate. The RMSE values between the preoperative design and the implanted outer contour of bone blocks were 0.41 ± 0.15 mm. The digital approach showed advantages in intuitiveness (9.3 ± 0.5), understanding (9.0 ± 0.5), and helpfulness (8.4 ± 1.1) according to surgeons' VAS scores. CONCLUSIONS: A digital workflow provided encouraging results, in terms of accuracy and efficacy, for horizontal bone augmentation. TRIAL REGISTRATION: This study was registered in the National Clinical Trials Registry in 16/02/2023 under the identification number ChiCTR2300068361.

Immediately or delayed sinus augmentation after pseudocyst removal: A randomized trial.

PURPOSE: To compare clinical and histological outcomes of sinus augmentation performed immediately or 3 months after pseudocyst removal through a prospective randomized controlled study. MATERIALS AND METHODS: In total, 33 sinus augmentation procedures were performed in 31 patients. Augmentation was performed either immediately after pseudocyst removal (one-stage intervention) or after 3 months (two-stage intervention). Six months postoperatively, bone specimens were harvested, and histomorphometric analysis was performed as primary outcome. Data were recorded and evaluated for implant survival rates, marginal bone resorption, complication rate, and patient-centered outcomes (visual analogue scale [VAS]). RESULTS: There were no baseline differences between groups or dropouts. Twelve biopsies obtained for histomorphometric analysis showed that delayed sinus augmentation, when compared to immediated led to a 1.1% increased mineralized bone ratio (95% confidence interval [CI]: -15.9 to 13.7). Graft leakage and acute sinusitis occurred in one patient in the one-stage group, none in the two-stage group. No pseudocyst recurrence was observed until the end of 1-year follow-up. Median VAS scores for overall acceptance were significantly increase of 1.4 (95% CI: 0.3-2.56) in immediate group. The degree of post-operative discomfort was not significantly different, although an increase of VAS (0.52, 95% CI: -0.32 to 1.37) was observed in delay group. CONCLUSIONS: Both procedures of sinus augmentation immediately and 3 months after pseudocyst removal could obtain comparable histological outcomes and had low complication rates. Patients who underwent the one-stage procedure had a short treatment course and high satisfaction rates, but this procedure is technically challenging to perform. This clinical trial was not registered prior to participant recruitment and randomization. The clinical trial registration number is ChiCTR2200063121. The hyperlink is as follows: https://www.chictr.org.cn/showproj.html?proj=172755.

Implant Survival and Complication Prevalence in Complete-Arch Implant-Supported Fixed Dental Prostheses: A Retrospective Study with a Mean Follow-up of 5 Years.

Purpose: To evaluate the implant survival and the prevalence of biologic and mechanical complications in edentulous patients restored with complete-arch implant-supported fixed dental prostheses (IFDPs). Materials and Methods: Patients restored with complete-arch screw-retained IFDPs between January 2012 and December 2019 with a minimum 2-year follow-up were included. Outcome measures were cumulative survival rate (CSR) for implants and prostheses, biologic complications, and mechanical complications. A generalized estimating equation model was used to estimate potential risk factors for mechanical complications. Patient satisfaction was investigated using a standardized questionnaire. Results: A total of 44 prostheses supported by 268 implants in 30 patients were included for a mean duration of 4.8 years (range: 2 to 9 years). Eighteen of the prostheses were zirconia-ceramic (group ZC), and 26 were titanium-ceramic (group TC). The CSR for the implants and IFDPs was 99.3% (95% CI: 98.2% to 100.3%) and 92.5% (95% CI: 84.2% to 100.8%), respectively. The most common biologic complication was peri-implant mucositis (4.5%), followed by peri-implantitis (3.0%). The most common mechanical complication was ceramic chipping (45.5%), followed by crown debonding (13.6%) and framework fracture (4.5%). There was no significant difference in the prevalence of complications between groups TC and ZC (P > .050). The presence of cantilever (OR = 5.54, P = .048) and maxillary arch (OR = 5.94, P = .041) were significantly associated with mechanical complications. Patient satisfaction scores were generally high, but some continued to be bothered by speech problems (13.6%). Conclusion: Complete-arch IFDPs presented reliable clinical outcomes for edentulous patients with a high implant survival rate and a high level of patient satisfaction. However, a high incidence of mechanical complications occurred in the long term.

Ubiquitin modification in osteogenic differentiation and bone formation: From mechanisms to clinical significance.

The ubiquitin-proteasome system is an important pathway for mediating posttranslational modification and protein homeostasis and exerts a wide range of functions in diverse biological processes, including stem cell differentiation, DNA repair, and cell cycle regulation. Many studies have shown that ubiquitination modification plays a critical role in regulating the osteogenic differentiation of stem cells and bone formation through various mechanisms. This review summarizes current progress on the effects and mechanisms of ubiquitin modification on transcription factors and signaling pathways involved in osteogenic differentiation. Moreover, the review highlights the latest advances in the clinical application of drugs in bone tissue engineering. A thorough understanding of ubiquitin modifications may provide promising therapeutic targets for stem cell-based bone tissue engineering.

Does a Severely Resorbed Subantral Ridge Decrease Long-Term Implant Survival Rate with Sinus Floor Augmentation?

PURPOSE: To identify the impact of residual bone height on 5-year implant survival and prosthetic complication rates in patients who underwent maxillary sinus grafting. MATERIALS AND METHODS: A total of 87 consecutive patients were treated with 104 lateral approach maxillary sinus floor augmentation procedures with 100% deproteinized bovine bone and received 169 implants. The analysis considered patient age, sex, time of implant placement, and residual bone height. Patients with < 3 mm residual bone height were assigned to the study group; otherwise, they were placed in the control group. RESULTS: The mean follow-up was 68.2 months (0 to 103 months). The mean residual bone height was 1.8 mm in the study group and 4.1 mm in the control group. The 5-year implant survival and prosthetic complication rates were, respectively, 97.4% and 8.0% in the study group and 100% and 12.5% in the control group. Residual bone height, sex, age, and time of implant placement were not significant factors for the 5-year implant survival or prosthetic complication rate. The lateral bone wall was significantly thinner in the study group. The grafted bone height reduction was significantly different at 6 months and 2 years postoperation in both groups, but there was no difference in the change in grafted bone height reduction over time between the two groups. CONCLUSION: A residual bone height < 3 mm did not impact the survival rates of implants placed in grafted maxillary sinuses or the prosthetic complication rate after 5 years of functional loading.

Influence of buccal emergence profile designs on peri-implant tissues: A randomized controlled trial.

BACKGROUND: The prosthetic emergence profile design might be an important factor in postsurgical mucosal recession etiology. Therefore, a restorative buccal emergence profile designed correctly might reduce gingival margin recession. PURPOSE: To compare the marginal gingival level and the width/height (W/H) ratio between two profile configurations of single implant-supported restorations at molar sites. MATERIALS AND METHODS: Twenty-one patients requiring a single mandibular molar tooth replacement with supracrestal mucosal thickness ≥2 mm were recruited and randomly assigned to a prosthesis buccal emergence profile design based on the buccal mucosal W/H ratio (Test Group) or maintained the original emergence profile of the healing abutment (Control Group). Assessments were made before delivery of the definitive restoration (T0), at prosthesis placement (T1), one (T2), and 12 (T3) months after loading. The gingival margin level change (△GM), initial emergence angle, buccal mucosal W/H ratio, marginal bone loss (MBL), implant failure, and complications were assessed. RESULTS: The gingival recession in the test group (0.13 ± 0.32 mm) was significantly lower than in the control group (0.63 ± 0.38 mm) at T3 (p = 0.006). The initial emergence angle in the test group (31.4 ± 7.22 degrees) was significantly lower than the control group (40.0 ± 7.60 degrees) (p = 0.025). The W/H ratio in the test group at T2 was significantly higher than at T0 but remained stable thereafter. The W/H ratio presented a continued rising trend in the control group. CONCLUSIONS: When the initial supracrestal soft tissue thickness was ≥2 mm, a restorative emergence profile based on the W/H ratio significantly reduced gingival margin recession. An emergence angle of 32.4 degrees showed better behavior in maintaining the gingival margin than 40 degrees. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR190002210.

Epithelial cell transforming factor ECT2 is an important regulator of DNA double-strand break repair and genome stability.

Proteins containing breast cancer type 1 (BRCA1) C-terminal domains play crucial roles in response to and repair of DNA damage. Epithelial cell transforming factor (epithelial cell transforming sequence 2 [ECT2]) is a member of the BRCA1 C-terminal protein family, but it is not known if ECT2 directly contributes to DNA repair. In this study, we report that ECT2 is recruited to DNA lesions in a poly (ADP-ribose) polymerase 1-dependent manner. Using co-immunoprecipitation analysis, we showed that ECT2 physically associates with KU70-KU80 and BRCA1, proteins involved in nonhomologous end joining and homologous recombination, respectively. ECT2 deficiency impairs the recruitment of KU70 and BRCA1 to DNA damage sites, resulting in defective DNA double-strand break repair, an accumulation of damaged DNA, and hypersensitivity of cells to genotoxic insults. Interestingly, we demonstrated that ECT2 promotes DNA repair and genome integrity largely independently of its canonical guanine nucleotide exchange activity. Together, these results suggest that ECT2 is directly involved in DNA double-strand break repair and is an important genome caretaker.

Knockdown of LAP2α inhibits osteogenic differentiation of human adipose-derived stem cells by activating NF-κB.

BACKGROUND: Lamina-associated polypeptide 2α (LAP2α) is a nucleoplasmic protein that has been involved in the regulation of the cell cycle, gene transcription, and adult stem cell function. LAP2α down-regulation is linked to age-related osteoporosis and bone deformities; however, the underlying mechanisms remain obscure. The present study aimed to elucidate the function of LAP2α in the osteogenic differentiation of human adipose-derived stem cells (hASCs), which are attractive sources for bone tissue engineering. METHODS: The expression of LAP2α during the osteogenic differentiation of hASCs was detected firstly. A loss of function investigation was then carried out to characterize the function of LAP2α in osteogenic differentiation of hASCs both in vitro and in vivo. Moreover, RNA-sequences, western blotting, and confocal analyses were performed to clarify the molecular mechanism of LAP2α-regulated osteogenesis. RESULTS: We found that LAP2α expression was upregulated upon osteogenic induction. Both in vitro and in vivo experiments indicated that LAP2α knockdown resulted in impaired osteogenic differentiation of hASCs. Mechanistically, we revealed that LAP2α deficiency activated nuclear factor kappa B (NF-κB) signaling by controlling the cytoplasmic-nuclear translocation of p65. CONCLUSIONS: Collectively, our findings revealed that LAP2α functions as an essential regulator for osteogenesis of hASCs by modulating NF-κB signaling, thus providing novel insights for mesenchymal stem cell-mediated bone tissue engineering.

Influence of crown-to-implant ratio and different prosthetic designs on the clinical conditions of short implants in posterior regions: A 4-year retrospective clinical and radiographic study.

BACKGROUND: Short implants (intra-bony length ≤ 8 mm) are generally considered as an alternative to bone augmentation in challenging situations; however, clinical evidence from large-scale studies with long follow-up regarding the application of short implants remains deficient. PURPOSE: The present study aimed to assess the mid-term clinical outcomes of short implants supporting fixed prostheses in the posterior region, and to investigate the effects of the crown-to-implant ratio (C/I), and other patient-, implant-, prosthesis-relevant factors on the clinical conditions around short implants. MATERIALS AND METHODS: 180 Thommen short implants in 130 partially edentulous patients were enrolled in the study after 3 to 7 (mean 4.2) years of follow-up. Potential risk factors (patient sex and age, implant diameter and location, splinted vs single-tooth restorations, retention mode, anatomical and clinical C/I ratios) were evaluated according to the following outcomes: Implant survival, marginal bone loss (MBL), and mechanical and biological complications. RESULTS: In total, four implants in four patients failed as a result of peri-implantitis. The cumulative survival rate was 97.8% for implant-based analysis. The peri-implant MBL around 180 short implants was 0.90 ± 0.78 mm. The mean clinical C/I ratio was 1.16 ± 0.36. Correlation analysis revealed that the influence of the clinical C/I ratio and patient age were significant for MBL (P < .05), whereas other potential risk factors showed no significant association with the outcome. Among 180 short implants, 24 cases (13.3%) had biological complications and 32 cases (17.8%) had mechanical complications, respectively. Peri-implant MBL and complication rates around splinted and non-splinted implants were not statistically different. CONCLUSION: Within the limitations of this study, short implants supporting fixed prostheses in the posterior region achieved predictable clinical outcomes over a 3 to 7 year period. Within the range of 0.47 to 3.01, the higher the C/I ratio, the less the peri-implant MBL.

Exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells promote bone regeneration.

OBJECTIVES: The present study aimed to investigate whether exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration. MATERIALS AND METHODS: Exosomes enriched with miR-375 (Exo [miR-375]) were generated from hASCs stably overexpressing miR-375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting. The construction efficiency of Exo (miR-375) was evaluated with qRT-PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR-375) was investigated with ALP and ARS assays. Furthermore, dual-luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR-375 in osteogenic regulation. Finally, Exo (miR-375) were embedded with hydrogel and applied to a rat model of calvarial defect, and µ-CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR-375) in bone regeneration. RESULTS: miR-375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR-375) at 50 µg/mL improved the osteogenic differentiation of hBMSCs. With miR-375 absorbed by hBMSCs, insulin-like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3'UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR-375). After incorporated with hydrogel, Exo (miR-375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR-375) enhanced the bone regenerative capacity in a rat model of calvarial defect. CONCLUSIONS: Taken together, our study demonstrated that exosomes derived from miR-375-overexpressing hASCs promoted bone regeneration.

Tissue-Engineered Bone Immobilized with Human Adipose Stem Cells-Derived Exosomes Promotes Bone Regeneration.

Exosomes, nanoscale extracellular vesicles functioning as cell-to-cell communicators, are an emerging promising therapeutic in the field of bone tissue engineering. Here, we report the construction and evaluation of a novel cell-free tissue-engineered bone that successfully accelerated the restoration of critical-sized mouse calvarial defects through combining exosomes derived from human adipose-derived stem cells (hASCs) with poly(lactic-co-glycolic acid) (PLGA) scaffolds. The exosomes were immobilized on the polydopamine-coating PLGA (PLGA/pDA) scaffolds under mild chemical conditions. Specifically, we investigated the effects of hASC-derived exosomes on the osteogenic, proliferation, and migration capabilities of human bone marrow-derived mesenchymal stem cells in vitro and optimized their osteoinductive effects through osteogenic induction. Furthermore, an in vitro assay showed exosomes could release from PLGA/pDA scaffold slowly and consistently and in vivo results showed this cell-free system enhanced bone regeneration significantly, at least partially through its osteoinductive effects and capacities of promoting mesenchymal stem cells migration and homing in the newly formed bone tissue. Therefore, overall results demonstrated that our novel cell-free system comprised of hASC-derived exosomes and PLGA/pDA scaffold provides a new therapeutic paradigm for bone tissue engineering and showed promising potential in repairing bone defects.

The ubiquitin ligase SCFFBXW7α promotes GATA3 degradation.

GATA3 is a key transcription factor in cell fate determination and its dysregulation has been implicated in various types of malignancies. However, how the abundance and function of GATA3 are regulated remains unclear. Here, we report that GATA3 is physically associated with FBXW7α, and FBXW7α destabilizes GATA3 through assembly of a SKP1-CUL1-F-box E3 ligase complex. Importantly, we showed that FBXW7α promotes GATA3 ubiquitination and degradation in a GSK3 dependent manner. Furthermore, we demonstrated that FBXW7α inhibits breast cancer cells survival through destabilizing GATA3, and the expression level of FBXW7α is negatively correlated with that of GATA3 in breast cancer samples. This study indicated that FBXW7α is a critical negative regulator of GATA3 and revealed a pathway for the maintenance of GATA3 abundance in breast cancer cells.

Biomaterial Cues Regulate Epigenetic State and Cell Functions-A Systematic Review.

Biomaterial cues can act as potent regulators of cell niche and microenvironment. Epigenetic regulation plays an important role in cell functions, including proliferation, differentiation, and reprogramming. It is now well appreciated that biomaterials can alter epigenetic states of cells. In this study, we systematically reviewed the underlying epigenetic mechanisms of how different biomaterial cues, including material chemistry, topography, elasticity, and mechanical stimulus, influence cell functions, such as nuclear deformation, cell proliferation, differentiation, and reprogramming, to summarize the differences and similarities among each biomaterial cues and their mechanisms, and to find common and unique properties of different biomaterial cues. Moreover, this work aims to establish a mechanogenomic map facilitating highly functionalized biomaterial design, and renders new thoughts of epigenetic regulation in controlling cell fates in disease treatment and regenerative medicine.

Protein deubiquitinase USP7 is required for osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potential, including osteogenesis. Although protein deubiquitinases have been linked to stem cell fate determination, whether protein deubiquitination contributes to lineage commitment during osteogenic differentiation of hASCs remains to be investigated. The objective of this study was to evaluate the effects of the ubiquitin specific protease 7 (USP7) on osteogenic differentiation of hASCs. METHODS: An osteocalcin promoter driven luciferase reporter system was established to initially discover the potential association between USP7 and hASC osteogenesis. To further characterize the function of USP7 in osteogenic differentiation of hASCs, a combination of in vitro and in vivo experiments were carried out through genetic depletion or overexpression of USP7 using a lentiviral strategy. Moreover, HBX 41,108, a cyanoindenopyrazine-derived deubiquitinase inhibitor of USP7, was utilized at different doses to further examine whether USP7 regulated osteogenic differentiation of hASCs through its enzymatic activity. RESULTS: We demonstrated that USP7 depletion was associated with remarkable downregulation of the reporter gene activity. Genetic depletion of USP7 by lentiviral RNAi markedly suppressed hASC osteogenesis both in vitro and in vivo, while overexpression of USP7 enhanced the osteogenic differentiation of hASCs. Notably, chemical blockade via the small molecular inhibitor HBX 41,108 could efficiently mimic the effects of USP7 genetic depletion in a dose-dependent manner. CONCLUSIONS: Taken together, our study revealed that protein deubiquitinase USP7 is an essential player in osteogenic differentiation of hASCs through its catalytic activity, and supported the pursuit of USP7 as a potential target for modulation of hASC-based stem cell therapy and bone tissue engineering.

The X-linked deubiquitinase USP9X is an integral component of centrosome.

The X-linked deubiquitinase USP9X has been implicated in multiple pathological disorders including malignancies and X-linked intellectual disability. However, its biological function and substrate repertoire remain to be investigated. In this study, we utilized the tandem mass tag labeling assay to identify USP9X-regulated proteins and revealed that the expression of multiple genes is altered in USP9X-deficient cells. Interestingly, we showed that USP9X promotes stabilization of centrosome proteins PCM1 and CEP55 through its catalytic activity. Remarkably, we demonstrated that USP9X is physically associated and spatially co-localized with PCM1 and CEP55 in the centrosome, and we revealed that either PCM1 or CEP55 loss resulted in impairment of USP9X centrosome localization. Moreover, we showed that USP9X is required for centrosome duplication, and this effect is dependent on its catalytic activity and its N-terminal module, which is responsible for physical association of USP9X with PCM1 and CEP55. Collectively, our experiments identified USP9X as an integral component of the centrosome where it functions to stabilize PCM1 and CEP55 and promote centrosome biogenesis.

[Establishing a luciferase reporter system to evaluate osteogenic differentiation potential of human adipose-derived stem cells].

OBJECTIVE: Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs) in vitro, and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vivo. METHODS: The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OC(pro)-Luc-Puro construct. Then, the OC(pro)-Luc-Puro construct together with three assistant vectors: pMDLg/pRRE, pRSV-REV, and pVSVG, were transiently transfected into HEK293T cells followed by viral supernatants collection, filtration and concentration. Next, the hASCs stably expressing luciferase reporter gene driven by osteocalcin promotor were created with the lentivirus carrying OC(pro)-Luc-Puro cassette under puromycin selection. The OC(pro)-Luc-hASCs were then cultured in the absence or presence of osteogenic differentiation medium. On the 7th and 14th days, after osteogenic induction, cellular extracts were collected and analyzed by luciferase reporter assay. Meanwhile, alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR) analysis of osteogenic associated genes osteocalcin (OC), runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) were used to assess the osteogenic differentiation ability of OC(pro)-Luc-hASCs. RESULTS: OC(pro)-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated. On the 7th and 14th days after osteogenic induction, the luciferase activity of the cellular extracts from OC(pro)-Luc-hASCs was dramatically increased. Consistently, the extracellular matrix mineralization, as shown by Alizarin red S (ARS) staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC, Runx2 and ALP, although to variable extent, was observed upon osteogenic differentiation. These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OC(pro)-Luc-hASCs. CONCLUSION: We established a luciferase reporter system that could be used to rapidly, quantitatively and specifically determine osteogenic differentiation ability of hASCs. Comparing with the traditional time-consuming methods, the system we generated here was highly effective. This system not only can be used to examine ostogenic differentiation of hASCs in a high throughput manner, but also provides a way to monitor ostogenic differentiation of cells in vivo.