Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene

Ning, Botao; Tang, Yongmin

doi:10.3892/ol.2012.557

Cited by 4 publications

(3 citation statements)

References 11 publications

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“…Unfortunately, we could not use them for the following three reasons: (i) in our hands, the transfection efficiency observed was poor [ 30 ]; (ii) these primary cells did not do well upon performing the scratch (wound) compounded by growing them in medium containing 2 % FBS; (iii) these cells do not support repeated passages under selection conditions [ 31 ]. In order to test the ability of gB to mediate cell migration, we utilized adherent HeLa cells [ 32 ] primarily because these cells have been extensively used to study cell migration compared to HMVEC-d, HFF and 293 cells [ 33 , 34 ].…”

Section: Resultsmentioning

confidence: 99%

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

2016

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BackgroundKaposi’s sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is not only expressed on the envelope of mature virions but also on the surfaces of cells undergoing lytic replication. Among herpesviruses, KSHV gB is the only glycoprotein known to possess the RGD (Arg-Gly-Asp) binding integrin domain critical to mediating cell attachment. Recent studies described gB to also possess a disintegrin-like domain (DLD) said to interact with non-RGD binding integrins. We wanted to decipher the roles of two individually distinct integrin binding domains (RGD versus DLD) within KSHV gB in regulating attachment of cells over cell migration.MethodsWe established HeLa cells expressing recombinant full length gB, gB lacking a functional RGD (gBΔR), and gB lacking a functionally intact DLD (gBΔD) on their cell surfaces. These cells were tested in wound healing assay, Transwell migration assay, and adhesion assay to monitor the ability of the RGD and DLD integrin recognition motifs in gB to mediate migration and attachment of cells. We also used soluble forms of the respective gB recombinant proteins to analyze and confirm their effect on migration and attachment of cells. The results from the above studies were authenticated by the use of imaging, and standard biochemical approaches as Western blotting and RNA silencing using small interfering RNA.ResultsThe present report provides the following novel findings: (i) gB does not induce cell migration; (ii) RGD domain in KSHV gB is the switch that inhibits the ability of DLD to induce cellular migration thus promoting attachment of cells.ConclusionsIndependently, RGD interactions mediate attachment of cells while DLD interactions regulate migration of cells. However, when both RGD and DLD are functionally present in the same protein, gB, the RGD interaction-induced attachment of cells overshadows the ability of DLD mediated signaling to induce migration of cells. Furthering our understanding of the molecular mechanism of integrin engagement with RGD and DLD motifs within gB could identify promising new therapeutic avenues and research areas to explore.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2173-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

2016

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“…The full length of LMP-1 gene (accession no. AAK30567) was amplified using RT-PCR and then cloned into vector plasmid using the PCR cloning technique ( 30 ). Briefly, total RNA from CHON-001 cells was isolated using an TRIzol ® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols.…”

Section: Methodsmentioning

confidence: 99%

LIM mineralization protein‑1 inhibits IL‑1β‑induced human chondrocytes injury by altering the NF‑κB and MAPK/JNK pathways

Liu

Tong

et al. 2021

Exp Ther Med

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Osteoarthritis (OA) is a common degenerative disease that is associated with the degradation of articular cartilage. Accumulating evidence has confirmed that LIM mineralization protein-1 (LMP-1) is an important agent of bone formation and has been shown to be osteoinductive in various types of disease. However, the underlying mechanisms of LMP-1 in the pathogenesis of OA remain unknown. The present study aimed to evaluate the role and potential mechanism of LMP-1 in IL-1β-stimulated OA chondrocytes. CHON-001 cells were transfected with pcDNA3.1-LMP-1, pcDNA3.1, negative control-small interfering (si)RNA or LMP-1 siRNA for 24 h and then induced by IL-1β for 12 h to establish an OA model in vitro . Cell viability, apoptosis and inflammatory cytokine (IL-6, IL-8 and TNF-α) release were assessed using MTT assay, ﬂow cytometry and ELISA, respectively. The expression levels of LMP-1, cleaved-caspase 3, phosphorylated (p)-p65, p65, p-JNK and JNK were analyzed using reverse transcription-quantitative PCR and western blotting. Overexpression of LMP-1 notably alleviated the IL-1β-induced inflammatory response in CHON-001 cells, as shown by increased cell viability, decreased apoptosis, suppressed expression of cleaved-caspase 3 and a decreased cleaved-caspase 3/caspase 3 ratio. Moreover, IL-1β-induced secretion of IL-6, IL-8 and TNF-α in CHON-001 cells; this was reversed by pcDNA3.1-LMP-1. However, knocking down LMP-1 expression exert opposite effects on the IL-1β-induced inflammatory response in CHON-001 cells, as evidenced by the decreased cell viability, increased apoptosis, enhanced expression of cleaved-caspase 3 and cleaved-caspase 3/caspase 3 ratio and enhanced secretion of IL-6, IL-8 and TNF-α observed. The present data demonstrated that LMP-1 siRNA notably inhibited LMP-1 expression, suppressed cell viability, promoted apoptosis and enhanced cleaved-caspase 3 expression and cleaved-caspase 3/caspase 3 ratio. In addition, LMP-1 siRNA promoted the release of inflammatory factors in CHON-001 cells. It was also found that pcDNA3.1-LMP-1 inhibited p-p65 and p-JNK expression, as well as decreasing the p-p65/p65 and p-JNK/JNK ratio. Nevertheless, there was no significant difference in the mRNA expression levels of p65 and JNK between the groups. Taken together, these findings indicated that overexpression of LMP-1 alleviated IL-1β-induced chondrocytes injury by regulating the NF-κB and MAPK/JNK pathways, suggesting that LMP-1 may be a valuable therapeutic agent for OA treatment.

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“…TSP50 expression in the stably transfected cells was verified by western blotting. The cell line transfected with pcDNA3.0-TSP50 was designated TSP50-o/e cells [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

Protumoral TSP50 Regulates Macrophage Activities and Polarization via Production of TNF-α and IL-1β, and Activation of the NF-κB Signaling Pathway

et al. 2015

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Testes-specific protease 50 (TSP50) is abnormally overexpressed in many kinds of cancers and promotes cell proliferation and migration. However, whether TSP50 can influence the tumor microenvironment, especially the function of immune cells in the microenvironment, remains largely unknown. We demonstrated that exposure to the conditioned medium from TSP50-overexpressing cells, or co-culture with TSP50-overexpressing cells, enhanced the cytokine production and phagocytic activities of macrophages, and induced M2b polarization. Further investigation showed that production of TNF-α and IL-1β was strongly induced by TSP50 in TSP50-overexpressing cells. TSP50-induced TNF-α and IL-1β were main factors that mediated the effects of TSP50-overexpressing cells on macrophages. The NF-κB pathway could be activated in macrophages upon the treatment of conditioned medium of TSP50-overexpressing cells and its activation is necessary for the observed effects on macrophages. Taken together, our results suggested that oncogenic TSP50 expressed in cells could activate surrounding macrophages and induce M2b polarization, partly through inducing TNF-α/ IL-1β secretion and subsequent NF-κB pathway activation. This implies a potential mechanism by which oncogene TSP50 regulates tumor microenvironment to support tumor development.

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Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene

Cited by 4 publications

References 11 publications

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

LIM mineralization protein‑1 inhibits IL‑1β‑induced human chondrocytes injury by altering the NF‑κB and MAPK/JNK pathways

Protumoral TSP50 Regulates Macrophage Activities and Polarization via Production of TNF-α and IL-1β, and Activation of the NF-κB Signaling Pathway

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