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human embryonic kidney cells transformed with the sv40 large t antigen cell line 293ft (Thermo Fisher)

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Thermo Fisher human embryonic kidney cells transformed with the sv40 large t antigen cell line 293ft
Human Embryonic Kidney Cells Transformed With The Sv40 Large T Antigen Cell Line 293ft, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+embryonic+kidney+cells+transformed+with+the+sv40+large+t+antigen+cell+line+293ft/human+embryonic+kidney+cells/pm40338742-279-11-25
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human embryonic kidney cells transformed with the sv40 large t antigen cell line 293ft - by Bioz Stars, 2026-10

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Article Title: Mechanobiological mechanism of cyclic stretch-induced cell columnarization.
Article Snippet: Plasmids or siRNAs were packaged in Lipofectamine 3000 Transfection Reagent (Thermo Fisher, Waltham, MO, USA). .. To generate high levels of viral package proteins (gag and pol), human embryonic kidney cells transformed with the SV40 large T antigen cell line 293FT (Thermo Fisher, Waltham, MO, USA) were used to produce lentivirus packaging shRNA. ..

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NV RNA replicates in 293FT cells. (A) Schematic drawing of NV genome RNA showing viral proteins encoded by each of the three ORFs. (B) Western blotting detection of VPg precursors and VP1 in NV RNA-transfected 293FT cells. Cells were transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. Note that mature VPg (20 kDa) was not detectable by Western blotting. Actin and a nonspecific protein band served as equal loading controls. (C) NV VP1 expression is RNA replication dependent. 293FT cells were transfected with in vitro-transcribed NV genomic RNA (NV IVT RNA) or stool-isolated NV RNA and incubated for 48 h. VPg precursors and VP1 in cell lysates were detected by Western blotting. VP1 was concentrated by immunoprecipitation (IP) prior to Western blotting. (D and E) Kinetics of expression of VP1 and NV RNA during NV RNA replication. 293FT cells were transfected with carrier RNA or NV RNA and incubated for 8, 24, 48, and 72 h. At each time point, cells were either lysed for IP and Western blotting of VP1 (D) or extracted for cellular RNA to quantify NV RNA by RT-qPCR (E). WB, Western blotting.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replicates in 293FT cells. (A) Schematic drawing of NV genome RNA showing viral proteins encoded by each of the three ORFs. (B) Western blotting detection of VPg precursors and VP1 in NV RNA-transfected 293FT cells. Cells were transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. Note that mature VPg (20 kDa) was not detectable by Western blotting. Actin and a nonspecific protein band served as equal loading controls. (C) NV VP1 expression is RNA replication dependent. 293FT cells were transfected with in vitro-transcribed NV genomic RNA (NV IVT RNA) or stool-isolated NV RNA and incubated for 48 h. VPg precursors and VP1 in cell lysates were detected by Western blotting. VP1 was concentrated by immunoprecipitation (IP) prior to Western blotting. (D and E) Kinetics of expression of VP1 and NV RNA during NV RNA replication. 293FT cells were transfected with carrier RNA or NV RNA and incubated for 8, 24, 48, and 72 h. At each time point, cells were either lysed for IP and Western blotting of VP1 (D) or extracted for cellular RNA to quantify NV RNA by RT-qPCR (E). WB, Western blotting.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Western Blot, Transfection, Incubation, Expressing, In Vitro, Isolation, Immunoprecipitation, Quantitative RT-PCR

NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Transfection, Incubation, Western Blot, Immunofluorescence, Staining

NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P < 0.05; **, P < 0.01, compared to results with BSA pretreatment. (B) 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 48 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. (C) Western blotting of NV VP1 and ISG56 in the same cell lysates from the experiment shown in panel B. NV VP1 was concentrated by IP prior to Western blotting. Actin and two nonspecific protein bands (marked by asterisks) served as equal loading controls. (D) Time course of 293FT-ISRE-Luc reporter assay. 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 8, 24, 48, and 72 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h before each time point. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. Note that the vertical axis is in logarithmic scale. pIC, poly(I·C).

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P < 0.05; **, P < 0.01, compared to results with BSA pretreatment. (B) 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 48 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. (C) Western blotting of NV VP1 and ISG56 in the same cell lysates from the experiment shown in panel B. NV VP1 was concentrated by IP prior to Western blotting. Actin and two nonspecific protein bands (marked by asterisks) served as equal loading controls. (D) Time course of 293FT-ISRE-Luc reporter assay. 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 8, 24, 48, and 72 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h before each time point. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. Note that the vertical axis is in logarithmic scale. pIC, poly(I·C).

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Luciferase, Activity Assay, Transfection, Incubation, Positive Control, Western Blot, Reporter Assay

Replication of plasmid-derived U201 RNA does not induce an IFN response. (A) Schematic drawing of U201 genome transcript RNAs expressed from plasmids. Note that the Δ4607 mutation causes a frameshift and premature termination of Pol, creating an untranslated region between ORF1 and ORF2. (B) Western blotting of U201 polymerase (Pol), p35 (Nterm), and p41 in 293FT cells transfected with U201 or U201-Δ4607 plasmid for 24 h. A nonspecific protein band served as an equal loading control. (C) Laser scanning confocal microscopy images of 293FT cells transfected with U201 or U201-Δ4607 plasmid for 24 h. Cells were labeled with mouse monoclonal antibody to U201 VPg (red) and guinea pig antibody to U201 VP1 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm. The merged images show that both plasmids express ORF1 polyproteins (VPg-positive), but RNA replication (VP1-positive) occurs in only one of the U201-transfected cells, not in U201-Δ4607-transfected cells. (D) IFN-β-Luc reporter assay. 293FT cells were transfected with IFN-β-Luc reporter in combination with vector, U201, or U201-Δ4607 plasmid for 24 h. As a positive control, vector-transfected cells were infected with SeV (40 HA/ml) for 18 h. Luciferase activity was first normalized to that of an internal RLuc transfection control and then normalized to that of vector-transfected cells and is presented as fold induction. (E and F) RLuc and IFN-β-Luc dual reporter assay. 293FT cells were transfected with IFN-β-Luc reporter in combination with vector, U201-RLuc, or U201-Δ4607-RLuc plasmid for 24 h. As a positive control, vector-transfected cells were infected with SeV (40 HA/ml) for 18 h. RLuc activity is presented as original relative light units/second (RLU/s). Luciferase activity was normalized to that of vector-transfected cells and is presented as fold induction. (G) Western blotting of ISG56 in the same cell lysates as used in the experiments shown in panels E and F. Actin served as an equal loading control.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: Replication of plasmid-derived U201 RNA does not induce an IFN response. (A) Schematic drawing of U201 genome transcript RNAs expressed from plasmids. Note that the Δ4607 mutation causes a frameshift and premature termination of Pol, creating an untranslated region between ORF1 and ORF2. (B) Western blotting of U201 polymerase (Pol), p35 (Nterm), and p41 in 293FT cells transfected with U201 or U201-Δ4607 plasmid for 24 h. A nonspecific protein band served as an equal loading control. (C) Laser scanning confocal microscopy images of 293FT cells transfected with U201 or U201-Δ4607 plasmid for 24 h. Cells were labeled with mouse monoclonal antibody to U201 VPg (red) and guinea pig antibody to U201 VP1 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm. The merged images show that both plasmids express ORF1 polyproteins (VPg-positive), but RNA replication (VP1-positive) occurs in only one of the U201-transfected cells, not in U201-Δ4607-transfected cells. (D) IFN-β-Luc reporter assay. 293FT cells were transfected with IFN-β-Luc reporter in combination with vector, U201, or U201-Δ4607 plasmid for 24 h. As a positive control, vector-transfected cells were infected with SeV (40 HA/ml) for 18 h. Luciferase activity was first normalized to that of an internal RLuc transfection control and then normalized to that of vector-transfected cells and is presented as fold induction. (E and F) RLuc and IFN-β-Luc dual reporter assay. 293FT cells were transfected with IFN-β-Luc reporter in combination with vector, U201-RLuc, or U201-Δ4607-RLuc plasmid for 24 h. As a positive control, vector-transfected cells were infected with SeV (40 HA/ml) for 18 h. RLuc activity is presented as original relative light units/second (RLU/s). Luciferase activity was normalized to that of vector-transfected cells and is presented as fold induction. (G) Western blotting of ISG56 in the same cell lysates as used in the experiments shown in panels E and F. Actin served as an equal loading control.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Plasmid Preparation, Derivative Assay, Mutagenesis, Western Blot, Transfection, Confocal Microscopy, Labeling, Reporter Assay, Positive Control, Infection, Luciferase, Activity Assay

NV and U201 RNA replication generates dsRNA. (A and B) Laser scanning confocal microscopy images of 293FT cells transfected with NV RNA for 48 h or transfected with U201 or U201-Δ4607 plasmid for 24 h, as indicated. Cells were labeled with antibodies to dsRNA (red) and NV VP1 or p48 (green) (A) or with antibodies to dsRNA (red), U201 VP1 (green), and U201 VPg (magenta) (B). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV and U201 RNA replication generates dsRNA. (A and B) Laser scanning confocal microscopy images of 293FT cells transfected with NV RNA for 48 h or transfected with U201 or U201-Δ4607 plasmid for 24 h, as indicated. Cells were labeled with antibodies to dsRNA (red) and NV VP1 or p48 (green) (A) or with antibodies to dsRNA (red), U201 VP1 (green), and U201 VPg (magenta) (B). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, Labeling

NV and GII.3 VLPs do not induce an IFN response. 293FT-ISRE-Luc reporter cells were treated with increasing doses (12 and 36 μg/ml) of NV or GII.3 (strain TCH-104) VLPs or with 100 μg/ml poly(I·C) (as a positive control) in the medium for 6 h. Luciferase activity was normalized to that of untreated cells and is presented as fold induction. pIC, poly(I·C).

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV and GII.3 VLPs do not induce an IFN response. 293FT-ISRE-Luc reporter cells were treated with increasing doses (12 and 36 μg/ml) of NV or GII.3 (strain TCH-104) VLPs or with 100 μg/ml poly(I·C) (as a positive control) in the medium for 6 h. Luciferase activity was normalized to that of untreated cells and is presented as fold induction. pIC, poly(I·C).

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Positive Control, Luciferase, Activity Assay

NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control. (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P < 0.05, compared to results with carrier RNA-transfected 293FT cells with IFN-α and IFN-β stimulation, respectively, and no antibody treatment.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control. (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P < 0.05, compared to results with carrier RNA-transfected 293FT cells with IFN-α and IFN-β stimulation, respectively, and no antibody treatment.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Neutralization, Western Blot, Transfection, Reporter Assay, Lysis, Luciferase, Activity Assay

NV RNA replication is not enhanced by neutralization of type III IFNs. (A) Western blotting of NV VP1 and VPg in 293FT cells pretreated with type III IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for dilutions of the antibodies. Actin served as an equal loading control. (B) 293FT-ISRE-Luc reporter assay of type III IFN neutralizing antibodies. The experimental procedure was essentially the same as that described in the legend of Fig. 5D, except that type III IFN neutralizing antibodies were used, and reporter cells were stimulated with 50 ng/ml IL-29. **, P < 0.01, compared to results in carrier RNA-transfected 293FT cells with IL-29 stimulation and no antibody treatment.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication is not enhanced by neutralization of type III IFNs. (A) Western blotting of NV VP1 and VPg in 293FT cells pretreated with type III IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for dilutions of the antibodies. Actin served as an equal loading control. (B) 293FT-ISRE-Luc reporter assay of type III IFN neutralizing antibodies. The experimental procedure was essentially the same as that described in the legend of Fig. 5D, except that type III IFN neutralizing antibodies were used, and reporter cells were stimulated with 50 ng/ml IL-29. **, P < 0.01, compared to results in carrier RNA-transfected 293FT cells with IL-29 stimulation and no antibody treatment.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Neutralization, Western Blot, Transfection, Reporter Assay

NV RNA replication is not enhanced by knockdown of MAVS or IRF3. (A) Western blotting to confirm knockdown of MAVS. (B) ISRE-Luc assay to functionally confirm knockdown of MAVS. 293FT and 293FT-shMAVS cells were transduced with ISRE-Luc lentiviral particles for 24 h and then mock or SeV infected (40 HA/ml) for 18 h. Luciferase activity was normalized to that of mock-infected 293FT cells and is presented as fold induction. (C) Western blotting of NV VP1 and MAVS in 293FT and 293FT-shMAVS cells transfected with carrier RNA (−) or NV RNA (+) for 48 h. (D) Western blotting to confirm knockdown of IRF3. (E) ISRE-Luc assay to functionally confirm knockdown of IRF3. 293FT and 293FT-shIRF3 cells were transduced with ISRE-Luc lentiviral particles for 24 h and then mock or poly(I·C) treated (100 μg/ml in the medium) for 6 h. Luciferase activity was normalized to that of mock-treated 293FT cells and is presented as fold induction. (F) Western blotting of NV VP1 and IRF3 in 293FT and 293FT-shIRF3 cells transfected with carrier RNA (−) or NV RNA (+) for 48 h. (G) Effect of IRF3 knockdown on the number of NV RNA replicating cells. 293FT and 293FT-shIRF3 cells seeded in 48-well plates were transfected with NV RNA for 48 h. Cells were fixed for IF staining with antibody to NV VP1. The number of VP1-positive cells per well was counted and is presented as mean values from multiple wells. *, P < 0.05; **, P < 0.01; ns, not statistically significant, compared to results in 293FT cells. Actin (A, C, D, and F) and a nonspecific protein band (marked by a circle in C and F) served as equal loading controls.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication is not enhanced by knockdown of MAVS or IRF3. (A) Western blotting to confirm knockdown of MAVS. (B) ISRE-Luc assay to functionally confirm knockdown of MAVS. 293FT and 293FT-shMAVS cells were transduced with ISRE-Luc lentiviral particles for 24 h and then mock or SeV infected (40 HA/ml) for 18 h. Luciferase activity was normalized to that of mock-infected 293FT cells and is presented as fold induction. (C) Western blotting of NV VP1 and MAVS in 293FT and 293FT-shMAVS cells transfected with carrier RNA (−) or NV RNA (+) for 48 h. (D) Western blotting to confirm knockdown of IRF3. (E) ISRE-Luc assay to functionally confirm knockdown of IRF3. 293FT and 293FT-shIRF3 cells were transduced with ISRE-Luc lentiviral particles for 24 h and then mock or poly(I·C) treated (100 μg/ml in the medium) for 6 h. Luciferase activity was normalized to that of mock-treated 293FT cells and is presented as fold induction. (F) Western blotting of NV VP1 and IRF3 in 293FT and 293FT-shIRF3 cells transfected with carrier RNA (−) or NV RNA (+) for 48 h. (G) Effect of IRF3 knockdown on the number of NV RNA replicating cells. 293FT and 293FT-shIRF3 cells seeded in 48-well plates were transfected with NV RNA for 48 h. Cells were fixed for IF staining with antibody to NV VP1. The number of VP1-positive cells per well was counted and is presented as mean values from multiple wells. *, P < 0.05; **, P < 0.01; ns, not statistically significant, compared to results in 293FT cells. Actin (A, C, D, and F) and a nonspecific protein band (marked by a circle in C and F) served as equal loading controls.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Western Blot, Transduction, Infection, Luciferase, Activity Assay, Transfection, Staining

MAVS is not degraded in NV RNA replicating cells. (A) Western blotting of MAVS in 293FT cells transfected with carrier RNA or NV RNA or mock or HAV infected (multiplicity of infection of 1) for 48 h. NV RNA replication and HAV infection were confirmed by Western blotting of NV VP1 and HAV VP1/precursors, respectively. Actin served as an equal loading control. (B) Laser scanning confocal microscopy images of 293FT cells infected with HAV (multiplicity of infection of 0.1) or transfected with NV RNA for 48 h. Cells were labeled with antibodies to MAVS (red) and HAV VP1 or NV VP1 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: MAVS is not degraded in NV RNA replicating cells. (A) Western blotting of MAVS in 293FT cells transfected with carrier RNA or NV RNA or mock or HAV infected (multiplicity of infection of 1) for 48 h. NV RNA replication and HAV infection were confirmed by Western blotting of NV VP1 and HAV VP1/precursors, respectively. Actin served as an equal loading control. (B) Laser scanning confocal microscopy images of 293FT cells infected with HAV (multiplicity of infection of 0.1) or transfected with NV RNA for 48 h. Cells were labeled with antibodies to MAVS (red) and HAV VP1 or NV VP1 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Western Blot, Transfection, Infection, Confocal Microscopy, Labeling

NV RNA replication does not block an SeV-induced IFN response. 293FT cells were transfected with NV RNA. At 30 h post-RNA transfection (hpt), cells were mock or SeV infected (4 HA/ml). At 48 hpt (18 hpi for SeV), cells were fixed and labeled with antibodies to ISG56 (red) and NV VP1 (green). Nuclei were counterstained with DAPI (blue). The laser scanning confocal microscopy images show that SeV-induced upregulation of ISG56 occurs in both NV VP1-positive and -negative cells. Scale bars, 10 μm.

Journal: Journal of Virology

Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

doi: 10.1128/JVI.01425-16

Figure Lengend Snippet: NV RNA replication does not block an SeV-induced IFN response. 293FT cells were transfected with NV RNA. At 30 h post-RNA transfection (hpt), cells were mock or SeV infected (4 HA/ml). At 48 hpt (18 hpi for SeV), cells were fixed and labeled with antibodies to ISG56 (red) and NV VP1 (green). Nuclei were counterstained with DAPI (blue). The laser scanning confocal microscopy images show that SeV-induced upregulation of ISG56 occurs in both NV VP1-positive and -negative cells. Scale bars, 10 μm.

Article Snippet: The 293FT cell line, derived from human embryonic kidney HEK293 cells transformed with the simian virus 40 (SV40) large T antigen, was purchased from Invitrogen.

Techniques: Blocking Assay, Transfection, Infection, Labeling, Confocal Microscopy

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human embryonic kidney cells transformed with the sv40 large t antigen cell line 293ft (Thermo Fisher)

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