Autophagy-Related Gene Expression Analysis of Wild-type and atg5 Gene Knockout Mouse Embryonic Fibroblast Cells Treated with Polyethylenimine

2014 ◽  
Vol 11 (9) ◽  
pp. 3002-3008 ◽  
Author(s):  
Chia-Wei Lin ◽  
Ming-Shiou Jan ◽  
Jung-Hua Steven Kuo
Keyword(s):  
Gene Expression ◽  
Knockout Mouse ◽  
Gene Expression Analysis ◽  
Gene Knockout ◽  
Mouse Embryonic Fibroblast ◽  
Fibroblast Cells ◽  
Related Gene ◽  
Wild Type ◽  
Gene Knockout Mouse ◽  
Embryonic Fibroblast

MDM2 Promotes Ubiquitin-Independent Degradation of HoxA9: Implications for MLL-Fusion Protein Mediated Transformation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1418-1418
Author(s):  
Huiwu Zhao ◽  
Shenghao Jin ◽  
Alan M. Gewirtz
Keyword(s):  
Half Life ◽  
Fusion Proteins ◽  
Wild Type Mouse ◽  
Mouse Embryonic Fibroblast ◽  
Fibroblast Cells ◽  
Wild Type ◽  
Functional Inactivation ◽  
Embryonic Fibroblast ◽  
Ubiquitylation Assay

Abstract Chromosome translocations involving the mixed lineage leukemia gene (MLL) are associated with aggressive lymphoid and myeloid leukemias. Inappropriate activation of crucial developmental genes has been proposed as one important mechanism for triggering leukemia by MLL-fusions. For example, it is known that the HoxA9 homeodomain protein, a key regulator of hematopoiesis and embryonic development, can be induced by expression of MLL-fusion proteins, and that this event is essential for MLL-dependent leukemogenesis. Recently, it has been suggested that functional inactivation of p53 by MLL-fusions may also contribute to this process. In this study, we explored the effect of p53 suppression by MLL-fusions on HoxA9 function. Using a variety of experimental strategies including co-immunoprecipitation, protein turnover assay and in vivo ubiquitylation assay, we found that the well-known p53 target MDM2 plays an important role in HoxA9 stability. To examine whether MDM2 is associated with HoxA9, Flag tagged MDM2 and HA tagged HoxA9 expression vectors were co-transfected into human HEK293T cells. MDM2 was then immunoprecipitated with anti-Flag beads. HoxA9 was readily detected in the MDM2 immnunocomplex, indicating that HoxA9 indeed interacts with MDM2. To study the functional role of the interaction between MDM2 and HoxA9, the effect of MDM2 on HoxA9 stability was next investigated. Increasing amounts of an MDM2 expression vector was co-transfected into HEK293T cells with an equal amount of a HoxA9 expression plasmid. Following the transfections, HoxA9 protein expression in the lysates from the different transfections was compared. MDM2 was found to promote the degradation HoxA9 in a dose-dependent manner. This effect was specific, since MDM2 did not interfere with Nup98-HoxA9 fusion protein expression in the control experiment. To further demonstrate the effect of MDM2 on HoxA9 stability, HoxA9 half-life was examined in wild type mouse embryonic fibroblast cells and MDM2 negative mouse embryonic fibroblast cells by the measuring HoxA9 protein turnover rate upon cycloheximide treatment. In wild type mouse embryonic fibroblast cells, the HoxA9 protein half-life is less then 30 minutes while HoxA9 protein half-life is about 3 hours in MDM2 knockout mouse embryonic fibroblast cells. Finally, to elucidate the molecular mechanism by which MDM2 promotes HoxA9 degradation, an in vivo ubiquitylation assay was used to test whether MDM2 is a ubiquitin E3 ligase for HoxA9. MDM2 was found to be incompetent in promoting the ubiquitylation of HoxA9. However, the proteasome inhibitor MG-132 could stabilize HoxA9 protein in HoxA9 transfected HEK293T cells. Taken together, these data suggest that MDM2 regulates HoxA9 stability by a ubiquitin-independent proteasome-dependent pathway. Furthermore, our findings indicate that indirect inhibition of MDM2 through functional inactivation of p53 by MLL-fusions might stabilize HoxA9 protein at post-translation level and this might be an important mechanism for the full functional activation of HoxA9 by MLL-associated fusion proteins.

scholarly journals Kilham Polyomavirus: Activation of Gene Expression and DNA Replication in Mouse Fibroblast Cells by an Enhancer Substitution

2001 ◽  
Vol 75 (21) ◽  
pp. 10015-10023 ◽  
Author(s):  
Shouting Zhang ◽  
Göran Magnusson
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
T Antigen ◽  
Mouse Fibroblast ◽  
Fibroblast Cells ◽  
Large T Antigen ◽  
Wild Type ◽  
3T3 Cells ◽  
Late Promoter ◽  
Substitution Mutant

ABSTRACT The Kilham strain of polyomavirus (KV) infects vascular endothelial cells in vivo (J. E. Greenlee, Infect. Immun. 26:705–713, 1979), but no permissive cell type for growth of the virus in vitro has been identified. The failure of KV DNA to replicate in mouse fibroblast cells after transfection suggested that viral gene expression had narrow cell specificity. A KV substitution mutant having a part of the regulatory region of KV DNA replaced with a segment of the polyomavirus transcriptional enhancer was constructed. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA associated with protein to form particles with the density of virions in CsCl equilibrium gradients. However, these particles were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of early and late promoter activities by luciferase reporter gene expression showed that the enhancer substitution had a moderate positive effect on early gene expression and a large effect on the expression of the late genes. KV large T antigen inhibited the activities of both the wild-type and the substitution mutant early promoter, whereas only the mutant late promoter was activated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiation of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of KV large T antigen. Together, our data demonstrate several differences between the two types of large T antigen in their interactions with cellular proteins.

CIP elicitors on the defense response of A. macrocephala and its related gene expression analysis

2020 ◽  
Vol 245 ◽  
pp. 153107
Author(s):  
Peifeng Zhang ◽  
Fang Zheng ◽  
Lei Chen ◽  
Xiaofang Lu ◽  
Wei Tian
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Defense Response ◽  
Gene Expression Analysis ◽  
Related Gene

Calcium hypochlorite on mouse embryonic fibroblast cells (NIH3T3) in vitro cytotoxicity and genotoxicity: MTT and comet assay

2020 ◽  
Vol 47 (7) ◽  
pp. 5377-5383
Author(s):  
Şehnaz Yilmaz ◽  
Oguz Yoldas ◽  
Aysin Dumani ◽  
Gizem Guler ◽  
Seda Ilgaz ◽  
...  
Keyword(s):  
Comet Assay ◽  
Mouse Embryonic Fibroblast ◽  
In Vitro Cytotoxicity ◽  
Fibroblast Cells ◽  
Calcium Hypochlorite ◽  
Embryonic Fibroblast

scholarly journals Slow growth and unstable ribosomal RNA lacking pseudouridine in mouse embryonic fibroblast cells expressing catalytically inactive dyskerin

FEBS Letters ◽  
2013 ◽  
Vol 587 (14) ◽  
pp. 2112-2117 ◽  
Author(s):  
Bai-Wei Gu ◽  
Jingping Ge ◽  
Jian-Meng Fan ◽  
Monica Bessler ◽  
Philip J. Mason
Keyword(s):  
Ribosomal Rna ◽  
Slow Growth ◽  
Mouse Embryonic Fibroblast ◽  
Fibroblast Cells ◽  
Embryonic Fibroblast
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