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  • Conditional mutations of beta-catenin and APC reveal roles for canonical Wnt signaling in lens differentiation. 19515997

    Previous studies indicate that the Wnt/beta-catenin-signaling pathway is active and functional during murine lens development. In this study, the consequences of constitutively activating the pathway in lens during development were investigated.To activate Wnt/beta-catenin signaling, beta-catenin (Catnb) and adenomatous polyposis coli (Apc) genes were conditionally mutated in two Cre lines that are active in whole lens (MLR10) or only in differentiated fibers (MLR39), from E13.5. Lens phenotype in mutant lenses was investigated by histology, immunohistochemistry, BrdU labeling, quantitative RT-PCR arrays, and TUNEL.Only intercrosses with MLR10 resulted in ocular phenotypes, indicating Wnt/beta-catenin signaling functions in lens epithelium and during early fiber differentiation. Mutant lenses were characterized by increased progression of epithelial cells through the cell cycle, as shown by BrdU labeling, and phosphohistone 3 and cyclin D1 labeling, and maintenance of epithelial phenotype (E-cadherin and Pax6 expression) in the fiber compartment. Fiber cell differentiation was delayed as shown by reduced expression of c-maf and beta-crystallin and delay in expression of the CDKI, p57(kip2). From E13.5, there were numerous cells undergoing apoptosis, and by E15.5, there was evidence of epithelial-mesenchymal transition with numerous cells expressing alpha-smooth muscle actin. Quantitative PCR analyses revealed large changes in expression of Wnt target genes (Lef1, Tcf7, T (Brachyury), and Ccnd1), Wnt inhibitors (Wif1, Dkk1, Nkd1, and Frzb) and also several Wnts (Wnt6, Wnt10a, Wnt8b, and Wnt11).These data indicate that the Wnt/beta-catenin pathway plays key roles in regulating proliferation of lens stem/progenitor cells during early stages of fiber cell differentiation.
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    Reference
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  • Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro. 20427756

    In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC(50)) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.
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    Reference
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  • Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling. 22388892

    Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.
    Document Type:
    Reference
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  • Degradation of p21Cip1 through anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20) ubiquitin ligase complex-mediated ubiquitylation is inhibited by ... 22045811

    Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

  • Zebrafish mnx1 controls cell fate choice in the developing endocrine pancreas. 21989909

    The vertebrate endocrine pancreas has the crucial function of maintaining blood sugar homeostasis. This role is dependent upon the development and maintenance of pancreatic islets comprising appropriate ratios of hormone-producing cells. In all vertebrate models studied, an initial precursor population of Pdx1-expressing endoderm cells gives rise to separate endocrine and exocrine cell lineages. Within the endocrine progenitor pool a variety of transcription factors influence cell fate decisions, such that hormone-producing differentiated cell types ultimately arise, including the insulin-producing beta cells and the antagonistically acting glucagon-producing alpha cells. In previous work, we established that the development of all pancreatic lineages requires retinoic acid (RA) signaling. We have used the zebrafish to uncover genes that function downstream of RA signaling, and here we identify mnx1 (hb9) as an RA-regulated endoderm transcription factor-encoding gene. By combining manipulation of gene function, cell transplantation approaches and transgenic reporter analysis we establish that Mnx1 functions downstream of RA within the endoderm to control cell fate decisions in the endocrine pancreas progenitor lineage. We confirm that Mnx1-deficient zebrafish lack beta cells, and, importantly, we make the novel observation that they concomitantly gain alpha cells. In Mnx1-deficient embryos, precursor cells that are normally destined to differentiate as beta cells instead take on an alpha cell fate. Our findings suggest that Mnx1 functions to promote beta and suppress alpha cell fates.
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    Reference
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    Multiple