Efficient and Reliable Transfection of Mouse Erythroleukemia Cells Using Cationic Lipids

Elnitski, Laura; Hardison, Ross C.

doi:10.1006/bcmd.1999.0257

Cited by 13 publications

(12 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…MEL cells are erythroid progenitor cells at the stage of proerythroblast, and these cells continue to differentiate upon induction with DMSO. 19 As shown in Figure S1, the qRT-PCR analysis reflected a large reduction for both R-globin and β-globin mRNA concentrations in MEL cells treated with 10, 25, 40, or 110 nm spherical nAg, or 45 nm plate-like nAg, at a concentration of 1 μg/mL (P < 0.001), compared to those in control cells. It should be noted that all types of nAg used here caused no decrease in cell viability for MEL cells at 1 μg/mL (data not shown).…”

Section: Resultsmentioning

confidence: 79%

Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells

et al. 2013

View full text Add to dashboard Cite

Due to its antimicrobial activity, nanosilver (nAg) has become the most widely used nanomaterial. Thus far, the mechanisms responsible for nAg-induced antimicrobial properties and nAg-mediated toxicity to organisms have not been clearly recognized. Silver (Ag) ions certainly play a crucial role, and the form of nanoparticles can change the dissolution rate, bioavailability, biodistribution, and cellular uptake of Ag. However, whether nAg exerts direct "particle-specific" effects has been under debate. Here we demonstrated that nAg exhibited a robust inhibition on RNA polymerase activity and overall RNA transcription through direct Ag binding to RNA polymerase, which is separated from the cytotoxicity pathway induced by Ag ions. nAg treatment in vitro resulted in reduced hemoglobin concentration in erythroid cells; in vivo administration of nAg in mice caused profound reduction of hemoglobin content in embryonic erythrocytes, associated with anemia in the embryos.Embryonic anemia and general proliferation deficit due to the significant inhibition on RNA synthesis, at least partially, accounted for embryonic developmental retardation upon nAg administration. To date, there is no conclusive answer to the sources of nAg-mediated toxicity: Ag ions or "particlespecific" effects, or both. We here demonstrated that both Ag ions and nAg particles simultaneously existed inside cells, demonstrating the "Trojan horse" effects of nAg particles in posing biological impacts on erythroid cells. Moreover, our results suggested that "particle-specific" effects could be the predominant mediator in eliciting biological influences on erythroid cells under relatively low concentrations of nAg exposure. The combined data highlighted the inhibitory effect of nAg on RNA polymerase activity through a direct reciprocal interaction.

show abstract

Section: Resultsmentioning

confidence: 79%

Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells

et al. 2013

View full text Add to dashboard Cite

show abstract

“…MEL, HEL, and K-562 erythroid cells were lipofected by using Tfx-50 (Promega) as described in ref. 30.…”

Section: Methodsmentioning

confidence: 99%

Modification of the erythroid transcription factor GATA-1 by SUMO-1

Collavin

Gostissa

Avolio

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.

show abstract

“…The plasmid DNAs were transiently transfected into K562 cells using the cationic lipid reagent Tfx50 (Promega) following the procedure as described in Elnitski and Hardison (1999) and Elnitski et al (2001). Briefly, 0.8 µg of plasmid containing firefly luciferase reporter and 0.008 µg of cotransfection control plasmid expressing Renilla luciferase were transfected in triplicate into 4 ‫ן‬ 10 5 cells at a 2:1 ratio (charge to mass) of Tfx50 to DNA.…”

Section: Transient Transfection and Expressionmentioning

confidence: 99%

Experimental validation of predicted mammalian erythroid cis-regulatory modules

et al. 2006

View full text Add to dashboard Cite

Multiple alignments of genome sequences are helpful guides to functional analysis, but predicting cis-regulatory modules (CRMs) accurately from such alignments remains an elusive goal. We predict CRMs for mammalian genes expressed in red blood cells by combining two properties gleaned from aligned, noncoding genome sequences: a positive regulatory potential (RP) score, which detects similarity to patterns in alignments distinctive for regulatory regions, and conservation of a binding site motif for the essential erythroid transcription factor GATA-1. Within eight target loci, we tested 75 noncoding segments by reporter gene assays in transiently transfected human K562 cells and/or after site-directed integration into murine erythroleukemia cells. Segments with a high RP score and a conserved exact match to the binding site consensus are validated at a good rate (50%–100%, with rates increasing at higher RP), whereas segments with lower RP scores or nonconsensus binding motifs tend to be inactive. Active DNA segments were shown to be occupied by GATA-1 protein by chromatin immunoprecipitation, whereas sites predicted to be inactive were not occupied. We verify four previously known erythroid CRMs and identify 28 novel ones. Thus, high RP in combination with another feature of a CRM, such as a conserved transcription factor binding site, is a good predictor of functional CRMs. Genome-wide predictions based on RP and a large set of well-defined transcription factor binding sites are available through servers at http://www.bx.psu.edu/.

show abstract

Efficient and Reliable Transfection of Mouse Erythroleukemia Cells Using Cationic Lipids

Cited by 13 publications

References 22 publications

Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells

Silver Nanoparticles Induced RNA Polymerase-Silver Binding and RNA Transcription Inhibition in Erythroid Progenitor Cells

Modification of the erythroid transcription factor GATA-1 by SUMO-1

Experimental validation of predicted mammalian erythroid cis-regulatory modules

Contact Info

Product

Resources

About