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• Research Paper Volume 12, Issue 8 pp 7411-7430

  PGC-1α activator ZLN005 promotes maturation of cardiomyocytes derived from human embryonic stem cells

  Relevance score: 9.770429

  Yanping Liu, Huajun Bai, Fengfeng Guo, Phung N. Thai, Xiaoling Luo, Peng Zhang, Chunli Yang, Xueqin Feng, Dan Zhu, Jun Guo, Ping Liang, Zhice Xu, Huangtian Yang, Xiyuan Lu

  Keywords: embryonic stem cells, cardiomyocyte maturation, peroxisome proliferator-activated receptor gamma coactivator 1α, ZLN005, metabolism

  Published in Aging on April 28, 2020

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  Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in biomedical applications. However, the immature state of cardiomyocytes obtained using existing protocols limits the application of hPSC-CMs. Unlike adult cardiac myocytes, hPSC-CMs generate ATP through an immature metabolic pathway—aerobic glycolysis, instead of mitochondrial oxidative phosphorylation (OXPHOS). Hence, metabolic switching is critical for functional maturation in hPSC-CMs. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolism, which may help promote cardiac maturation during development. In this study, we investigated the effects of PGC-1α and its activator ZLN005 on the maturation of human embryonic stem cell-derived cardiomyocyte (hESC-CM). hESC-CMs were generated using a chemically defined differentiation protocol and supplemented with either ZLN005 or DMSO (control) on differentiating days 10 to 12. Biological assays were then performed around day 30. ZLN005 treatment upregulated the expressions of PGC-1α and mitochondrial function-related genes in hESC-CMs and induced more mature energy metabolism compared with the control group. In addition, ZLN005 treatment increased cell sarcomere length, improved cell calcium handling, and enhanced intercellular connectivity. These findings support an effective approach to promote hESC-CM maturation, which is critical for the application of hESC-CM in disease modeling, drug screening, and engineering cardiac tissue.

  

  The expression of PGC-1α was upregulated during cardiomyocyte differentiation; ZLN005 increased PGC-1α mRNA and protein level in hESC-CMs. (A) The relative mRNA and (B) protein expression of PGC-α during cardiomyocyte differentiation (mRNA, n=7; protein, n=5). (C) Schematic representation of the experimental schedule including hESC culture, cardiomyocyte differentiation, culture and treatment. (D) Effect of ZLN005 on mRNA levels (n=6). (E) Effect of ZLN005 on PGC-α protein expression (n=12).

  
  
  

  

  ZLN005 improved mitochondrial maturation of hESC-CMs. (A) qRT-PCR analysis of mitochondrial oxidative phosphorylation markers in hESC-CMs (n=7). (B) Representative lifetime profiles from control (black trace) and ZLN005-treated (red trace) hESC-CMs. (C) Basal oxygen consumption rates (lifetime slope) in control and ZLN005-treated hESC-CMs (n=8). (D) qRT-PCR analysis of mitochondrial biogenesis markers in hESC-CMs (n=7). (E) Mitochondrial DNA content, as determined by qRT-PCR using primers for mt-ND1 normalized to housekeeping gene β-actin (n=8). (F) Transmission electron microscopy (TEM) pictures in control and ZLN005-treated hESC-CMs. Scale bar, 2μm. (G) Basal ATP levels in hESC-CMs (n=6). A ratiometric analysis was performed to determine changes in the Lifetime fluorescence signal: Lifetime (μs) [T] = (D2-D1)/ln(W1/W2), where D is delay; W is fluorescence window value at each time point.

  
  
  

  

  ZLN005 improved cardiac structural maturation. (A) Representative immunostaining of α-ACTININ (red) and Hoechst 33342 (blue) in control- or ZLN005-treated hESC-CMs. Scale bar, 20μm and 5μm. (B and C) ZLN005-treated hESC-CMs showed significant increase in sarcomere length and a decrease in circularity index compared to control. n=20-60 cells per condition. (D) qRT-PCR analysis of cardiac structural maturation markers in control and ZLN005-treated hESC-CMs (n=6).

  
  
  

  

  ZLN005 treatment increased expression of Connexin 43 (CX43) and improved electrical activity in hESC-CMs. (A) Representative immunostaining images of CX43 (green) in control and ZLN005-treated hESC-CMs. Hoechst 33342 (blue) and α-ACTININ (red) were also co-stained in the same cells. Scale bar, 20 μm. (B) qRT-PCR analysis of CX43 expression (n=6). (C) Representative Western blot and quantification showed up-regulation of CX43 protein expression with ZLN005 treatment (n=6-9). (D) Representative color map of electrical signal propagation from control and ZLN005-treated hESC-CMs. The color map shows that the electrical signal is initiated at the upper right corner (red) and is propagated to the bottom left corner (blue). The black arrows indicate the direction of the instantaneously local electrical propagation. (E, F) Representative field potential tracings recorded from control (black) and ZLN005-treated (red) hESC-CMs, respectively. (G) Bar graph to compare field potential amplitude (FPA) between control and ZLN005-treated hESC-CMs (n=10). (H) Bar graphs to compare beating rate (left) and coefficient of variation (right) between control and ZLN005-treated hESC-CMs (n=10). (I) Bar graphs to compare inter-spike interval (left) and coefficient of variation (right) between control and ZLN005-treated hESC-CMs (n=10).

  
  
  

  

  ZLN005-treated hESC-CMs exhibited more negative resting membrane potential compared with control. (A) Representative spontaneous action potential traces from control (red trace) and ZLN005-treated (black trace) hESC-CMs. (B–E) Action potential properties of control and ZLN005-treated hESC-CMs: the resting membrane potentials (B), peak amplitude (C), velocity of upstroke (D), action potential durations at 90% repolarization (APD90) (E) n=24-54 cells for each group.

  
  
  

  

  ZLN005-treated hESC-CMs displayed an increase in calcium signaling and kinetics compared with control. (A) qRT-PCR analysis of cardiac calcium handling markers in control and ZLN005-treated hESC-CMs (n=6). (B) Representative intracellular Ca²⁺ transients from control (red trace) and ZLN005-treated (black trace) hESC-CMs. Calcium transients were evaluated by loading the hESC-CMs with fura-2 AM. (C–G) Ca²⁺ transients properties of control and ZLN005-treated ESC-CMs: The amplitude of Ca²⁺ transient (C) time to peak (D) maximal velocity of upstroke (E) decay time (F) maximal velocity of decay (G). n=15-20 cells for each group.

  
  
  

• Research Paper Volume 8, Issue 10 pp 2324-2336

  A comprehensive transcriptomic analysis of differentiating embryonic stem cells in response to the overexpression of Mesogenin 1

  Relevance score: 12.545365

  Dahai Liu, Qiang Zhang, Hong Zhang, Ling Tang, Wei Li, Dongming Zhang, Guoying Wu, Shoudong Ye, Qian Ban, Kan He

  Keywords: somitogenesis, microarray, pathway, embryonic stem cells, Msgn1

  Published in Aging on October 6, 2016

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  The mutation of somitogenesis protein Mesogenin 1 (Msgn1) has been widely used to study the direct link between somitogenesis and the development of an embryo. Several studies have used gene expression profiling of somitogenesis to identify the key genes in the process, but few have focused on the pathways involved and the coexpression patterns of associated pathways. Here we employed time-course microarray datasets of differentiating embryonic stem cells by overexpressing the transcription factor Msgn1 from the public database library of Gene Expression Omnibus (GEO). Then we applied gene set enrichment analysis (GSEA) to the datasets and performed candidate transcription factors selection. As a result, several significantly regulated pathways and transcription factors (TFs), as well as some of the specific signaling pathways, were identified during somitogenesis under Msgn1 overexpression, most of which had not been reported previously. Finally, significant core genes such as Hes1 and Notch1 as well as some of the TFs such as PPARs and FOXs were identified to construct coexpression networks of related pathways, the expression patterns of which had been validated by our following quantitative real-time PCR (qRT-PCR). The results of our study may help us better understand the molecular mechanisms of somitogenesis in mice at the genome-wide level.

  

  The comparison between related ChIP-seq results with our GSEA target genes.

  
  
  

  

  The summary of significantly regulated pathways based on GSEA under Msgn1 overexpression. The Venn diagram showed the comparisons of each time point. There are 100 most significant pathways at 12 hour, 113 significant pathways at 24 hour and 163 significant pathways at 48 hour. By the comparison of 3 different time points, 39 significantly associated pathways were overlapped. By the comparison of 12h and 24h time points, 51 significant pathways were overlapped. Similarly, 79 and 90 overlapping significant pathways were identified respectively while comparing between 12h and 48h, between 24h and 48h.

  
  
  

  

  The coexpression networks of related pathways during somitogenesis. We analyzed the signaling pathways network of dynamic regulation during somitogenesis at three different time points including12h, 24h and 48h. Fc epsilon RI signaling pathway was only significantly regulated in 12h, PPAR signaling pathway and Insulin signaling pathway were significantly regulated in 24h, Jak-STAT signaling pathway and B cell receptor signaling pathway were significantly regulated in 48h. 2 signaling pathways played roles at both 12h and 24h. Erb signaling pathway can activate Neurotrophin signaling pathway via MAPK signaling pathway. Jun, regulated by MAPK signaling pathway, induce neurotrophin activation. 5 signaling pathways regulated significantly at 24h and 48h, TGF-β signaling pathway activated Wnt signaling pathway via the expression of TGF-β1. Wnt1 is target gene of Wnt signaling pathway and Wnt1 can induced the activation of mTOR signaling pathway. 3 signaling pathways regulated significantly at 12h and 48h. 9 signaling pathways regulated at three different time points. MAPK and Hedgehog signaling pathways can activate Notch signaling pathway by activating Akt2 and Gli2 respectively. Notch1 and Hes1 are the downstream effectors of Notch signaling pathway and Hes1 is the target gene of Notch1. Hes7 can inhibit Notch target genes, so we hypothesize that Hes1 may have the similar effect. For simplicity, several target genes, gene products, and regulatory interactions are not shown.

  
  
  

  

  The expression patterns of regulated genes in major signaling pathways during somitogenesis under Msgn1 overexpression. Microarray analysis of of gene expression in the absence and presence of Dox over a 48 hour timecourse. log2 ratios of the normalized expression levels of Hes1, Notch1, Tgfβ1, Wnt1, Akt2 and Dli2 are presented. The expression of Hes1 and Tgfβ1 was upregulated under Msgn1 overexpression over a 48 hour timecourse. Notch1 expression was variable, presumably reflecting the dynamic expression of a cyclic gene, but was generally elevated by Msgn1 overexpression. Tgfβ1 expression was upregulated in treated groups compared with untreated ones at 24h, while Wnt1 expression was just opposite. It may confirm that TGF-β signaling pathway could activate Wnt signaling pathway via the expression of Tgfβ1. Notch1 and Hes1 expressions were both upregulated in the treated groups compared with untreated ones from 12h to 48h. The expression of Akt2 and Dli2 were both downregulated compared with control group while Notch1 expression was upregulated in treated groups, presumably reflecting other signaling pathways, Wnt signaling pathway for example, activate Notch signaling pathway. Here error bars indicate standard deviation of 3 biological replicates.

  
  
  

  

  The validation results of qRT-PCR. (A) Flag-tagged Msgn1 was introduced into 46C mESCs and the protein level of Flag-tagged Msgn1 was determined by Western blot. β-actin was used as a loading control. (B) qRT-PCR analysis of the indicated gene expression for the indicated time in mESCs and mESCs-derived EBs. Data represent mean±s.d. of three biological replicates. #p < 0.05 vs PB:Day 0. *p < 0.05, **p<0.01 vs PB-Mgsn1:Day 0.

  
  
  

• Review Volume 4, Issue 12 pp 878-886

  Embryonic stem cells and inducible pluripotent stem cells: two faces of the same coin?

  Relevance score: 9.301604

  Francesco Romeo, Francesco Costanzo, Massimiliano Agostini

  Keywords: embryonic stem cells, inducible pluripotent stem cells, cell-based therapy, self-renewal and differentiation

  Published in Aging on December 11, 2012

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  Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocysts and are characterized by the ability to renew themselves (self-renewal) and the capability to generate all the cells within the human body. In contrast, inducible pluripotent stem cells (iPSCs) are generated by transfection of four transcription factors in somatic cells. Like embryonic stem cells, they are able to self-renew and differentiate. Because of these features, both ESCs and iPSCs, are under intense clinical investigation for cell-based therapy. In this review, we revisit stem cell biology and add a new layer of complexity. In particular, we will highlight some of the complexities of the system, but also where there may be therapeutic potential for modulation of intrinsic stem cells and where particular caution may be needed in terms of cell transplantation therapies.

  

  (A) The stemness of ESCs is maintained by intrinsic (i.e. SOX2, NANOG and OCT4) and by extrinsic pathways (i.e. LIF, BMP4 and FGF). MicroRNAs also play a role in the maintenance of stem cells, and some are expressed during self-renewal (miR-269, miR-290-295 cluster, miR-371, miR-200c) while others are up-regulated during differentiation (miR-21, miR-22, miR-29, miR-134, miR-296, miR-470) (see text for details). (B) Role of p53 in the maintenance of genomic stability in ESCs. During DNA damage, p53 is activated (via Ser315 phosphorylation) and binds the Nanog promoter to repress its expression. The outcome of p53 activation is to induce the differentiation of ESCs into other cell types that they can go into a senescent state or induces apoptosis to preserve genome stability.

  
  
  

  

  (A) iPSCs are generated by the transduction in somatic cells of “pluripotency” factors. The resulting iPSCs have similar properties to ESCs. (B) p53 pathways regulate the efficiency of reprogramming. Overexpression of the oncogenic c-myc during reprogramming induces the ARF/p53 pathway that drives the cells to apoptosis or senescence and the miR-34 family negatively regulates the expression of Nanog and Sox2. In a miR-34 null-context, the expression of Nanog and Sox2 is increased, resulting in a higher reprogramming efficiency.

  
  
  

  

  Bone marrow transplantation is widely employed in the clinic for several diseases including cancer and haematological disorders. More recently, MSCs are stepping in. They could be used to treat inflammatory conditions such as Multiple Sclerosis and Pulmonary Fibrosis. The employment of iPSCs in cell-based therapy is a relative new tool. However, because their potential tumorigenicity in the clinical setting remains to be clarified, perhaps iPSCs should stay on the bench meanwhile and be used in drug screening and human disease models.

  
  
  

• Research Perspective Volume 3, Issue 10 pp 920-933

  Roles of FGF signaling in stem cell self-renewal, senescence and aging

  Relevance score: 10.154199

  Daniel L. Coutu, Jacques Galipeau

  Keywords: fibroblast growth factor, self-renewal, senescence, aging, adult stem cells, embryonic stem cells

  Published in Aging on October 9, 2011

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  The aging process decreases tissue function and regenerative capacity, which has been associated with cellular senescence and a decline in adult or somatic stem cell numbers and self-renewal within multiple tissues. The potential therapeutic application of stem cells to reduce the burden of aging and stimulate tissue regeneration after trauma is very promising. Much research is currently ongoing to identify the factors and molecular mediators of stem cell self-renewal to reach these goals. Over the last two decades, fibroblast growth factors (FGFs) and their receptors (FGFRs) have stood up as major players in both embryonic development and tissue repair. Moreover, many studies point to somatic stem cells as major targets of FGF signaling in both tissue homeostasis and repair. FGFs appear to promote self-renewing proliferation and inhibit cellular senescence in nearly all tissues tested to date. Here we review the role of FGFs and FGFRs in stem cell self-renewal, cellular senescence, and aging.

  

  In normal proliferating human cells, telomeres at the end of chromosomes are shortened at every cell division unless the cells express telomerase. When telomeres get too short, genomic instability ensues and a DNA-damage response under the control of the p21 pathway is induced. This causes growth arrest and intrinsic cellular senescence. Transduction of these senescence cells with a telomerase construct reverses this growth arrest and leads to immortalization. When cells undergo stress (e.g. reactive oxygen species, ionizing radiations, etc.) they can undergo p16-mediated extrinsic senescence even though they possess long telomeres. Re-expression of telomerase in this case does not rescue this irreversible growth arrest. Murine cells have very long telomeres and are not thought to be susceptible to intrinsic senescence in normal conditions. However, they are very sensitive to extrinsic senescence. Murine cells often escape from p16-mediated senescence and get immortalized but the mechanism for this is unclear.

  
  
  

  

  A) FGFRs possess three extracellular immunoglobulin-like domains (Ig I to III), a transmembrane domain (TM) and an intracellular protein tyrosine kinase domain (PTK).The third Ig-like domain (III) is thought to confer ligand specificity. The C-terminal half of this IgIII domain (dotted line) is alternatively encoded by either exon 8 or 9 of the receptor gene, which create the two main isoforms of FGFR1, 2 and 3 (IIIb for exon 8 and IIIc for exon 9). Other isoforms also exist (no PTK domain, no TM domain) but are less abundant. B) Creation of the ternary complex between heparan sulfate proteoglycans (HSPGs), FGF ligands, and FGFRs leads to autophosphorylation of the PTK domains and activation of a number of intracellular pathways downstream. FRS2 and Grb2 and the main mediators of the signaling and activate various effectors such as PI3K/AKT and MAPKs. Other pathways (Shp2, PLC-γ) are also activated. Note that activation of the PI3K pathway can lead to phosphorylation of MDM2 on Ser186, leading to its translocation to the nucleus and subsequent degradation of p53.

  
  
  

• Research Perspective Volume 3, Issue 2 pp 162-167

  SirT1 brings stemness closer to cancer and aging

  Relevance score: 11.458712

  Vincenzo Calvanese, Mario F. Fraga

  Keywords: Sirtuin, pluripotency, epigenetics, senescence, self-renewal, differentiation, embryonic stem cells

  Published in Aging on February 9, 2011

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  Sirtuin 1 acts in various cell processes, deacetylating both chromatin and non-histone proteins, and its role in cancer and aging has long been studied and debated. Here we discuss another aspect of SirT1 biology, its function as a stem cell pluripotency and differentiation regulator. We evaluate the implications of these findings in sirtuin inhibition-based cancer treatment and in the application of sirtuin activation for anti-aging therapy.

  

  For each process, the two main effects in which SirT1 has been implicated are indicated in the external circle.

  
  
  

• Research Paper Volume 2, Issue 7 pp 415-431

  miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues

  Relevance score: 10.832255

  Laura R. Saunders, Amar Deep Sharma, Jaime Tawney, Masato Nakagawa, Keisuke Okita, Shinya Yamanaka, Holger Willenbring, Eric Verdin

  Keywords: SIRT1; mouse embryonic stem cells; miRNAs; differentiation; post-transcriptional regulation; reprogramming

  Published in Aging on July 17, 2010

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  SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD⁺ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.

  

  (A-B) Protein and RNA were extracted from mESC and tissues from ~6-week-old mice. (A) Western blot analysis with antibodies against SIRT1 (Frye antiserum top blot; Upstate antiserum lower blot), HDAC1, HDAC2, and tubulin. (B) qRT-PCR analysis of SIRT1, HDAC1, and HDAC2 normalized to GAPDH levels. Data are mean ± s.d. for four samples. (C-D) Protein and RNA were isolated from mESCs differentiated in vitro for up to 20 days (EBs d2-20). (C) Western blots analysis of expression of SIRT1, various HDACs, markers of pluripotent embryonic stem cells, and markers of differentiation. Data are representative of four experiments. (D) qRT-PCR analysis of SIRT1, HDAC2, markers of pluripotent embryonic stem cells, and markers of differentiation. Data were normalized to GAPDH and plotted as expression relative to the mean of four mESC samples. Data are mean ± s.d. for four samples.

  
  
  

  

  (A) mESCs were differentiated and treated on d8 with the proteasome inhibitor MG-132 (10 μM, 3-7 h), and protein lysates were analyzed on western blots. Data are representative of four experiments. (B) Protein levels of SIRT1 and REST relative to tubulin levels were quantified by densitometry with NIH Image. (C-E) The consequences of Dicer inactivation and loss of small RNAs were assessed in protein lysates and RNA from livers of control and Dicer^flox/flox mice injected with the AAV8 vector expressing cre at the indicated times. (C) Western blotting was used to analyze 70 μg of liver lysate and 10 μg of mESC lysate. (D) SIRT1 protein levels relative to tubulin or GAPDH were quantified by densitometry. (E) SIRT1 and Dicer mRNA levels were measured by qRT-PCR. Data are mean ± s.d. for four samples. (F-H) Lung fibroblasts were cultured from Dicer^Flox/Flox mice and infected with adenoviral Cre or GFP. (F) SIRT1 protein levels were measured by western blotting 72 h after Cre inactivation of Dicer. (G) SIRT1 protein levels relative to tubulin were quantified by densitometry. (H) mRNA levels of SIRT1 and Dicer were measured by qRT-PCR. Data are mean ± s.d. for three samples. (I-K) siRNAs were transfected into NIH3T3 cells to knockdown DGCR8, Dicer, or GL3 luciferase as a control. (I) DGCR8 knockdown and increased SIRT1 protein levels were analyzed by western blotting 72 h after siRNA transfection. Data are representative of three experiments. (G) qRT-PCR analysis confirmed Dicer knockdown and no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for three samples.

  
  
  

  

  (A) 18 miRNAs from nine miRNA families that potentially target the 3'-UTR of SIRT1 were induced during mESC differentiation at the time SIRT1 protein was downregulated. Their fold induction in d20 embryoid bodies above their expression in undifferentiated mESCs was plotted on the y-axis, and the location of their seed binding site in the 3'-UTR of mSIRT1 was plotted on the x-axis. (B-C), qRT-PCR of miRNA expression relative to miR-16 from undifferentiated mESCs and differentiating embryoid bodies of specific miRNAs that potentially target SIRT1. Data are mean ± s.d. for four samples.

  
  
  

  

  (A) Luciferase assays were performed 24 h after transfection of the full-length 1.6 kb SIRT1 3'-UTR downstream of luciferase (SIRT1 3'-UTR) or constructs with 4 bp in the seed-binding regions mutated (SIRT1 3'-UTR 181mt, left panel; SIRT1 3'-UTR 9mt, right panel) and control, miR-181a, b, and c miRNA mimics (left panel) or pSuper and pSuper miR-9 expression constructs (right panel). Data are mean ± s.d. for eight experiments. (B-C) mESCs were transfected with individual miRNA expression constructs; protein and RNA were isolated 48 h later. (B) Repression of SIRT1 protein was analyzed by western blotting. Data are representative of six experiments. (C) qRT-PCR analysis of SIRT1 mRNA levels and mature miRNA levels. Data are mean ± s.d. for four samples.

  
  
  

  

  (A-C) mESCs were differentiated and transfected at d4 and d7 with LNA probes. Protein and RNA were isolated on indicated days. (A) qRT-PCR of miR-9 shows the expected upregulation during differentiation and 35% inhibition when embryoid bodies were transfected with LNA-miR-9 but not with LNA-SCR. (B) qRT-PCR show no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for four samples and representative of three experiments. (C) Western blot analysis shows that the downregulation of SIRT1 protein during mESC differentiation was specifically inhibited in cells transfected with LNA-miR-9 but not by transfection of LNA-SCR or untransfected controls. Data are representative of four experiments. (D-F) EBs were dissociated and transfected at d6 with LNA probes. Protein and RNA were isolated on d11. (D) Western blot analysis shows upregulation of SIRT1 protein in EBs transfected with LNA-miR-9 but not LNA-SCR. (E) qRT-PCR analysis shows inhibition of miR-9 in EBs transfected with LNA-miR-9, but not with LNA-SCR, and no significant change in SIRT1 mRNA levels (F). Data are mean ± s.d. for four samples.

  
  
  

  

  (A) mESCs, MEFs, and iPS cells were subject to western blot analysis with antibodies to the indicated proteins. (B) SIRT1, HDAC1, and HDAC2 protein levels relative to tubulin were quantified by densitometry. (C) qRT-PCR analysis of SIRT1 mRNA levels in mESCs, MEFs, and iPS cells were measured relative to GAPDH. (D) qRT-PCR analysis of miRNA expression relative to miR-16 in mESCs, MEFs, and iPS. Data are mean ± s.d. for three samples.

  
  
  

• Research Paper pp undefined-undefined

  Zbtb34 promotes embryonic stem cell proliferation by elongating telomere length

  Relevance score: 11.980509

  Zheng Liu, Xinran Wei, Yue Gao, Xiaodie Gao, Xia Li, Yujuan Zhong, Xiujuan Wang, Chong Liu, Tianle Shi, Jiabin Lv, Tao Liu

  Keywords: Zbtb34, zinc finger, telomere, Pot1b, mouse embryonic stem cells

  Published in Aging on Invalid Date

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  Zbtb34 is a novel zinc finger protein, which is revealed by biological software analysis to have 3 zinc fingers, but its functions remain unknown. In this study, mouse Zbtb34 cDNA was amplified by PCR and inserted into the plasmid pEGFP-N1 to generate Zbtb34-EGFP fusion protein. The upregulation of Zbtb34 in mouse embryonic stem cells promoted telomere elongation and increased cell proliferation. In order to understand the above phenomena, the telomere co-immunoprecipitation technique was employed to investigate the relationship between Zbtb34 and telomeres. The results indicated that Zbtb34 could bind to the DNA sequences of the telomeres. Alanine substitution of the third zinc finger abolished such binding. Since Pot1 is the only protein binding to the single-stranded DNA at the end of the telomeres, we further investigated the relationship between Zbtb34 and Pot1. The results revealed that the upregulation of Zbtb34 decreased the binding of Pot1b to the telomeres. Through the upregulation of Pot1b, the binding of Zbtb34 to the telomeres was also reduced. In conclusion, we showed that the main biological function of Zbtb34 was to bind telomere DNA via its third ZnF, competing with Pot1b for the binding sites, resulting in telomere elongation and cell proliferationt.