• Biomedical Science Letters
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• v.11 no.3
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• pp.327-332
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• 2005

Sf21 cells become popular as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike human glucose transporters, very little is known about the characteristics of the endogenoussugar transporter(s) in Sf21 cells. Thus, some kinetic properties of the sugar transport system were investigated, involving the uptake of 2-deoxy-D-glucose (2dG1c). In order to obtain a true measure of the initial rate of uptake, the uptake of $[^3H]2dGlc$ from both low $(100{\mu}M)$ and high (10 mM) extracellular concentrations was measured over periods ranging from 30 sec to30 min. The data obtained indicated that the uptake was linear for at least 2 min at both concentrations, suggesting that measurements made over a 1min time course would reflect initial rates of the jexpse uptake. To determine $K_m\;and\;V_{max}$ of the endogenous glucose transporter(s) in Sf21 cells, the uptake of 2dG1c was measured over a range of substrate concentrations $(50{\mu}M\~10mM)$ 2dG1c uptake by the Sf21 cells appeared to involve both saturable and non-saturable (or very low affinity) components. A saturable transport system for 2dG1c was relatively high, the $K_m$ value for uptake being < 0.45 mM. The $V_{max}$ value obtained for 2dG1c transport in the Sf21 cells was about 9.7-folds higher than that reported for Chinese hamster ovary cells, which contain a GLUT1 homologue. Thus, it appeared that the transport activity of the Sf21 cells was very high. In addition, the Sf21 glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1485-1490
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• 2006

In this study, comparative replicational properties of Hyphantria cunea nucleopolyhedrovirus (HycuNPV) in Lymantria dispar (IPLB-LdElta) and Spodoptera frugiperda (IPLB-Sf21) cell lines were investigated. Our microscopic observations showed that cytopathic effects (CPEs) in LdElta cells appeared 12 h later than those in Sf21 cells. Whereas polyhedral inclusion bodies (PIBs) formed at 48 h postinfection (p.i.) in LdElta cells, it formed at 36 h p.i. in Sf21 cells. Extracellular virus production determined according to the 50% tissue culture infective dose ($TCID_{50}$) method in LdElta cells started about 12 h later when compared with Sf21 cells. Titers of extracellular virus in LdElta and Sf21 cells were calculated as $1.77{\times}10^9$ plaque forming units (PFU)/ml and $5.6{\times}10^9PFU/ml$, respectively, at 72 h p.i. We also showed that viral DNA replication began at 12 h p.i. in both cell lines. Viral protein synthesis was determined by SDS-polyacrylamide gel electrophoresis (PAGE) and polyhedrin synthesis was observed at 12 h p.i. in both cell lines. The results indicate that while the synthesis of macromolecules is 12 h later and production of extracellular virus is almost 3-fold lower in LdElta cells compared with those in Sf21 cells, the LdElta cell line is still a productive cell line for infection of HycuNPV.

• Biomedical Science Letters
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• v.12 no.1
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• pp.17-22
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• 2006

The baculovirus expression system is a powerful method for producing large amounts of the human erythrocyte-type glucose transport protein, heterologously. Characterization of the expressed protein is expected to show its ability to transport sugars directly. To achieve this, it is a prerequisite to know the properties of the endogenous sugar transport system in Spodoptera frugiperda Clone 21 (Sf21) cells, which are commonly employed as a host permissive cell line to support the baculovirus replication. The Sf21 cells can grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transport system. However, unlike the human glucose transport protein that has a broad substrate and inhibitor specificity, very little is known about the nature of the endogenous sugar transport system in Sf21 cells. In order to characterize further the inhibitor recognition properties of the Sf21 cell transporter, the ability of phloretin, cytochalasin B and D-fructose to inhibit 2-deoxy-D-glucose (2dGlc) transport was examined by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. The 2dGlc transport in the Sf21 cells was very potently inhibited by phloretin, the aglucone of phlorizin with a $K_i$ similar to the value of about $2{\mu}M$ reported for inhibition of glucose transport in human erythrocytes. However, the Sf21 cell transport system was found to differ from the human transport protein in being much less sensitive to inhibition by cytochalasin B (apparent $K_i$ approximately $10\;{\mu}M$). In contrast, It is reported that the inhibitor binds the human erythrocyte counterpart with a $K_d$ of approximately $0.12\;{\mu}M$. Interestingly, the Sf21 glucose transport system also appeared to have high affinity for D-fructose with a $K_i$ of approximately 5mM, contrasting the reported $K_m$ of the human erythrocyte transport protein for the ketose of 1.5M.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.161-166
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• 1998

To study the usefulness of Se301 cells, which is originated from Spodoptera exigua and has susceptibility to the Autographa californica NPV (AcNPV), as a host for the AcNPV-based expression vector system, we compared the characteristics of AcNPV in Se301 and Sf-21 cells. The symptom by viral infection was similar in both of cells, but the ratio of polyhedra released from the cell was higher in Se301 cells than in Sf-21 cells. The overall PIB productivity of AcNPV was similar in both cells but the size of polyhedra was larger in Se301 cells. While the polyhedrin expression efficiency was about 2.4 times higher in Se301 cells than in Sf-21 cells, the viral growth was higher in Sf-21 cells. These results suggested that Se301 cell is very useful in the AcNPV-based expression system as a host.

• Journal of Microbiology
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• v.45 no.2
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• pp.133-138
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• 2007

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via ${\beta}-galactosidase$ expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.

• Molecules and Cells
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• v.45 no.10
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• pp.718-728
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• 2022

Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The in vivo expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.

• Biomedical Science Letters
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• v.11 no.4
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• pp.487-492
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• 2005

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. Spodoptera frugiperda Clone 21 (Sf2l) cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, very little is known about the properties of the endogenous sugar transporter(s) in Sf2l cells, although a saturable transport system for hexose uptake has been previously revealed in the Sf cells. In order to further examine the substrate and inhibitor recognition properties of the Sf2l cell transporter, the ability of hexoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. Transport was effectively inhibited by D-mannose and D-glucose. Of the hexoses tested, L-glucose had the least effect on 2dGlc transport in the Sf2l cells, indicating that the transport is stereoselective. Unlike the human HepG2 type glucose transport system, D-mannose had a somewhat greater affinity for the Sf2l cell transporter than D-glucose, implying that the hydroxyl group at the C-2 position is not necessary for strong binding. However, epimerization at the C-4 position of D-glucose (D-galactose) resulted in a dramatic decrease in affinity of the hexose for the Sf2l cell transporter. Such a lowering of affinity might be the result of the involvement of the C-4 hydroxyl in hydrogen bonding. It is therefore suggested that Sf2l cells were found to contain an endogenous sugar transport activity that in several aspects resembles the human HepG2 type glucose transporter, although the insect and human transporters do differ in their affinity for cytochalasin B.

• Biomedical Science Letters
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• v.9 no.4
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.361-364
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• 1999

The binding characteristics of Anagrapha falcifera nuclear polyhedrosis virus (AtNPV) to Spodoptera frugiperda 21 (Sf21) cells were investigated. The cells displayed an affinity of 4.7×10/sup 10/M/sup -1/ with about 3,300 binding sites per cell. The biochemical nature of the AfNPV-binding sites on the cell surface was also partially identified. Our findings suggest that the binding-site moiety has a glycoprotein component, but that the direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that AfNPV entry into Sf21 cells may be via receptor-mediated endocytosis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.142-146
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• 2003

Three insect cell lines, Sf9, Sf21 and Tn5Bl-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was a ppropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5Bl-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3 times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammoniumion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line. respectively. The maximum specific ${\beta}$-galactosidase production rate was 4.5 fold that of the Sf9 cell line, a 3 times higher protease activity per cell.