scholarly journals HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells

2019 ◽  
Vol 40 (4) ◽  
Author(s):  
Andrew J. Kueh ◽  
Samantha Eccles ◽  
Leonie Tang ◽  
Alexandra L. Garnham ◽  
Rose E. May ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Adhesion ◽  
Dna Replication ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cells ◽  
Human Cell Lines ◽  
Hela Cell Lines ◽  
Mouse Tissues ◽  
Sirna Knockdown

ABSTRACT HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.

scholarly journals Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2825 ◽  
Author(s):  
Heidi A. Neubauer ◽  
Stuart M. Pitson
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Polyclonal Antibody ◽  
Human Cell ◽  
Sphingosine Kinase ◽  
Immunofluorescence Staining ◽  
Human Cell Lines ◽  
Sphingosine Kinase 2 ◽  
Hela Cell Lines ◽  
Rabbit Polyclonal Antibody

Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout (Sphk2-/-) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

Altered gene expression during cellular aging

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 386-389 ◽  
Author(s):  
James R. Smith ◽  
Olivia M. Pereira-Smith
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Cellular Senescence ◽  
Human Cell ◽  
Messenger Rna ◽  
Human Cells ◽  
Cellular Aging ◽  
Human Cell Lines ◽  
Normal Human

The limited division potential of normal human diploid fibroblasts in culture represents a model system for cellular aging. Observations indicate cellular senescence is an active process. Senescent cells, although unable to divide, are actively metabolizing. Hybrids from fusion of normal and immortal human cells exhibit limited division potential, suggesting that the phenotype of cellular senescence is dominant and supporting the hypothesis that senescence is genetically programmed. Fusion of immortal human cell lines with each other has identified four complementation groups for indefinite division. This indicates that a limited number of specific genes or processes are involved in senescence. Senescent cells express highly abundant DNA synthesis inhibitory messenger RNAs and produce a surface membrane associated protein inhibitor of DNA synthesis not expressed in young cells. Senescent cell membranes were used as immunogen to generate three monoclonal antibodies reacting specifically with senescent but not young cells in several normal human cell lines. We have also found that fibronectin messenger RNA accumulates to high levels in senescent cells. The role of these changes in gene expression in senescence is being explored.Key words: cellular senescence, human cells.

Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines

2007 ◽  
Vol 90 (1) ◽  
pp. 37-41 ◽  
Author(s):  
R.M. Kern ◽  
Z. Yang ◽  
P.S. Kim ◽  
W.W. Grody ◽  
R.K. Iyer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Mouse Tissues ◽  
Sodium Phenylbutyrate

Interferon potentiates cytotoxic effects of 5-fluorouracil on cell proliferation of established human cell lines originating from neoplastic tissues☆

1983 ◽  
Vol 17 (3) ◽  
pp. 239-247 ◽  
Author(s):  
Toshihiro Miyoshi ◽  
Shigeo Ogawa ◽  
Toshinori Kanamori ◽  
Masahiro Nobuhara ◽  
Masayoshi Namba
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Human Cell ◽  
Human Cell Lines ◽  
Cytotoxic Effects ◽  
Neoplastic Tissues

scholarly journals Relative cytotoxicity of complexes of platinum(II) and palladium(II) against pure cell culture Paramecium caudatum and human cell lines A431 and HaCaT

2018 ◽  
Vol 7 (1) ◽  
pp. 28-38 ◽  
Author(s):  
Aleksei Vladimirovich Eremin ◽  
Roman Vladimirovich Suezov ◽  
Polina Sergeevna Grishina ◽  
Alexander Ivanovich Ponyaev ◽  
Nicolay Leonidovich Medvedskiy
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Mtt Assay ◽  
Human Cell ◽  
Human Cells ◽  
Epidermoid Carcinoma ◽  
Binuclear Complexes ◽  
Human Cell Lines ◽  
Paramecium Caudatum ◽  
Pure Cell

The results of cytotoxicity cis-diamine mono- and binuclear complexes of platinum(II) and palladium(II) are presented. The cytotoxicity was investigated by the method of biotesting with Paramecium caudatum and by MTT-assay with human cells: epidermoid carcinoma A431 and minimal transformed aneuploid keratinocytes HaCaT. Cytotoxicity of complexes towards protists is higher than against human cells, however, comparatively, HaCaT is more sensitive than A431 by the treatment all complexes. It is noted that cytotoxicity of palladium(II) complexes is higher than the analogues with platinum(II).

scholarly journals Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells

2021 ◽  
Vol 22 (21) ◽  
pp. 11805
Author(s):  
Rasmus Ree ◽  
Karoline Krogstad ◽  
Nina McTiernan ◽  
Magnus E. Jakobsson ◽  
Thomas Arnesen
Keyword(s):  
Catalytic Activity ◽  
Cell Lines ◽  
Human Cell ◽  
Hypoxia Inducible Factor ◽  
Human Cells ◽  
Human Proteome ◽  
Human Cell Lines ◽  
Structural Studies ◽  
Hypoxia Inducible Factor 1Α ◽  
Lysine Acetyltransferase

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.

scholarly journals Gene knockout shows that PML (TRIM19) does not restrict the early stages of HIV-1 infection in human cell lines

2017 ◽  
Author(s):  
Nasser Masroori ◽  
Pearl Cherry ◽  
Natacha Merindol ◽  
Jia-xin Li ◽  
Caroline Dufour ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Gene Knockout ◽  
Human Cells ◽  
Restriction Factor ◽  
Type I ◽  
Human Cell Lines ◽  
Early Stages ◽  
Human T Cell ◽  
Hiv 1

AbstractThe PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. By contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNAi with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used CRISPR-Cas9 to knock out its gene in epithelial, lymphoid and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Over-expression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages.ImportancePML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early post-entry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

scholarly journals Gene Knockout Shows That PML (TRIM19) Does Not Restrict the Early Stages of HIV-1 Infection in Human Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nasser Masroori ◽  
Pearl Cherry ◽  
Natacha Merindol ◽  
Jia-xin Li ◽  
Caroline Dufour ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Gene Knockout ◽  
Human Cells ◽  
Restriction Factor ◽  
Type I ◽  
Human Cell Lines ◽  
Early Stages ◽  
Human T Cell ◽  
Hiv 1

ABSTRACTThe PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. In contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNA interference, with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used the clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9) system to knock out its gene in epithelial, lymphoid, and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Overexpression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5α (another human TRIM protein), and TRIM5α was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages.IMPORTANCEPML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early postentry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

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