Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in the IRF3−/− MEFs. (A) MEFs derived from wild-type (WT) and IRF3−/−, IRF7−/−, or MyD88−/− mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B) MEFs derived from WT and IRF3−/− mice were inoculated with rBV-GFP at an MOI of 100, incubated at 4°C for 30 min, and washed three times with PBS. The total cellular DNA was extracted, and the amounts of baculovirus genome were quantified by real-time PCR. Data represent means ± SD from 2 independent experiments. (C) MEFs derived from WT and IRF3−/− mice were inoculated with 2 doses of rBV-GFP (MOI of 100 or 20). At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. (D) MEFs derived from WT and IRF3-deficient mice were inoculated with 2 doses of rBV-GFP (MOI of 100 and 20). At 24 h after inoculation, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, IRF3, or β-actin, respectively.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, SDS Page

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: IRF3-dependent enhancement of gene transduction by recombinant baculovirus. (A) Cell extracts of MEFs derived from WT or IRF3−/− mice and FLAG-mIRF3-transduced IRF3−/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IRF3, or β-actin, respectively. (B) MEFs derived from WT or IRF3−/− mice and FLAG-mIRF3-transduced IRF3-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent the means ± SD from 3 independent experiments. (C) MEFs were inoculated with rBV-luc at an MOI of 100. At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. IRF3 (green) was stained with the appropriate antibodies, followed by staining with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained by DAPI.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, SDS Page, Luciferase, Activity Assay, Staining

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus vectors through the STING/TBK1/IRF3 axis in MEFs. (A) MEFs derived from wild-type (WT) and STING- or ZBP1-deficient mice were inoculated with rBV-GFP (MOI of 100). At 24 h after inoculation, the GFP signal was determined by microscopic observation after fixation in 4% paraformaldehyde. (B and C) MEFs derived from WT and STING- or ZBP1-deficient mice were inoculated with rBV-luc (MOI of 100) or rBV-GFP (MOI of 100). At 24 h after inoculation, the luciferase activity of cell lysates was determined (B), and the production of IFN-β in the culture supernatant was determined by sandwich ELISA (C). (D) The MEFs were inoculated with wild-type baculovirus (AcNPV) (MOI of 100). At 6 h after inoculation, cells were fixed in 50% methanol–50% acetone for 10 min. STING (green) and ER-calnexin (red) were stained with the appropriate antibodies, followed by staining with Alexa Fluor 488- or Alexa Fluor 555-conjugated second antibodies, respectively. Nuclei were stained by DAPI.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Luciferase, Activity Assay, Sandwich ELISA, Staining

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: RLR signaling pathways participate in the suppression of gene transduction in MEFs upon infection with recombinant baculovirus. (A) MEFs derived from wild-type (WT) and IPS-1-, TBK1-, ZBP1-, or IRF3-deficient mice were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments. (B) MEFs derived from WT and IPS-1−/− mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, GFP expression was detected by microscopic observation after fixation in 4% paraformaldehyde. (C) MEFs derived from WT or IPS-1-deficient mice were inoculated with rBV-GFP at an MOI of 100. At 24 h after inoculation, total RNA was extracted, and the expression of GFP, IFN-β, and IP-10 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Cell extracts of MEFs derived from WT or IPS-1−/− mice and FLAG-mIPS-1-transduced IPS-1−/− MEFs were subjected to SDS-PAGE and immunoblotted with antibodies against FLAG, IPS-1, or β-actin, respectively. (E) MEFs derived from WT or IPS-1−/− mice and FLAG-mIPS-1-transduced IPS-1-deficient MEFs were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity was determined. Data represent means ± SD from 3 independent experiments.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Protein-Protein interactions, Transduction, Infection, Recombinant, Derivative Assay, Luciferase, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, SDS Page

Journal: Journal of Virology

Article Title: Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

doi: 10.1128/JVI.03055-13

Figure Lengend Snippet: Efficient gene transduction by recombinant baculovirus in HCV replicon-harboring cells. (A) Huh7OK1 cells (cured) and the HCV replicon-harboring cells derived from genotype 1a (RMT strain), 1b (con1 strain), and 2a (JFH1 strain) were inoculated with rBV-luc (MOI of 100) or VSV-luc at an MOI of 5 and (B) with rBV-luc at MOI of 5, 10, 25, and 50. At 24 h after inoculation, the luciferase activity of cell lysates was determined. (C) Huh7 cells were inoculated with rBV-GFP (MOI of 100) or VSV-GFP (NCP mutant) at an MOI of 0.05. At 24 h after inoculation, total RNA was extracted, and the expression of IP-10, ISG15, and IL-8 mRNAs was determined by real-time PCR. Data from the real-time PCR were normalized to the amount of GAPDH mRNA. (D) Huh7 cells infected with HCVcc at an MOI of 1 and incubated for 72 h were inoculated with rBV-GFP (MOI of 100) in the presence or absence of human recombinant IFN-α (rIFN-α) (100 U/ml). At 24 h after inoculation, the cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies against GFP, NS5A, or β-actin, respectively. (E) Relative luciferase activity in Huh7 cells with IRF3, IPS-1, or STING knocked down. Cells were inoculated with rBV-luc at an MOI of 100. At 24 h after inoculation, the luciferase activity of cell lysates was determined (right panel). Luciferase activity is normalized to control shNC cells, and data represent the means ± SD from 2 independent experiments. Total RNA was extracted, and the expression of IRF3, IPS-1, or STING mRNA was determined by real-time PCR (left panel). Data from the real-time PCR were normalized to the amount of GAPDH mRNA.

Article Snippet: The human IRF-3, IPS-1, and STING genes were amplified by using the TaqMan probes (IRF3, Hs01547283_m1; IPS-1, Hs00920075_m1; STING, Hs00736958).

Techniques: Transduction, Recombinant, Derivative Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Infection, Incubation, SDS Page, Control

Journal: Cellular & molecular immunology

Article Title: The zinc-finger protein ZFYVE1 modulates TLR3-mediated signaling by facilitating TLR3 ligand binding.

doi: 10.1038/s41423-019-0265-6

Figure Lengend Snippet: Fig. 2 ZFYVE1 is important for TLR3-mediated signaling. a Effects of ZFYVE1-RNAi on the expression of ZFYVE1. As shown in the upper two panels, HEK293 cells (1 × 105) were transfected with HA-ZFYVE1 (0.1 μg), HA-β-actin (0.01 μg), and the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 24 h before immunoblotting analysis. As shown in the lower two panels, HEK293 cells (1 × 105) were transfected with the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 12 h. The cells were then selected with puromycin (1 μg/mL) for 24 h before immunoblotting analysis. b Effects of ZFYVE1-RNAi on poly(I:C)-induced activation of the IFN-β promoter, ISRE, and NF-κB. 293-TLR3 cells (1 × 105) were transfected with the indicated reporter and ZFYVE1-RNAi plasmids (0.5 μg) for 36 h and then were or were not treated with poly(I:C) (20 μg/mL) for 6 h before luciferase assays. c Effects of ZFYVE1-RNAi on the poly(I:C)-induced transcription of downstream genes. 293-TLR3 cells (1 × 105) were transfected with control or ZFYVE1-RNAi plasmid (1 μg) for 12 h. The cells were selected with puromycin (1 μg/mL) for 24 h and then were or were not treated with poly(I:C) (50 μg/mL) for 3 h before qPCR analysis. d Effects of ZFYVE1 deficiency on the poly(I:C)-induced transcription of downstream genes. ZFYVE1-deficient HT1080 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control HT1080 cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) for the indicated times before qPCR analysis. e Effects of ZFYVE1 deficiency on the LPS- induced transcription of downstream genes. ZFYVE1-deficient 293-TLR4 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control 293-TLR4 cells (1 × 105) were or were not treated with LPS (100 ng/mL) for 2 h before qPCR analysis. f Effects of ZFYVE1 deficiency on the poly(I:C)- and LPS-induced phosphorylation of TBK1, IRF3, and p65. ZFYVE1-KO and control cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before immunoblotting analysis. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test)

Article Snippet: Reagents, antibodies, and cells The following reagents, antibodies, and cells were purchased from the indicated companies: TRIzol (TaKaRa Bio); SYBR Green (BioRad); a dual-specific luciferase assay kit (Promega); polybrene (Millipore); poly(I:C), PGN, and R848 (Invivogen); LPS and DNase I (Sigma); EZ-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), a first strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), a cell mitochondria isolation kit (Beyotime), a nuclear and cytoplasmic Cellular & Molecular Immunology _#####################_ extraction kit (Applygen); ELISA kits to detect murine IFN-β, TNFα, and IL-6 (BioLegend); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (Origene), p-IRF3 (Cell Signaling Technology), p-p65 (Cell Signaling Technology), TLR3 (Cell Signaling Technology), p-Tyr (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), AIF (Santa Cruz Biotechnology), KDEL (Santa Cruz Biotechnology), p65 (Abcam), TBK1 (Abcam), p-TBK1 (Abcam), TRIF (Abcam), LMNB1 (Proteintech), β-tubulin (Life Technology), ZFYVE1 and RAB5A (Abclonal); and Alexa Fluor 488- and Alexa Fluor 594-conjugated goat anti-mouse IgG antibodies (Invitrogen).

Techniques: Expressing, Transfection, Western Blot, Activation Assay, Luciferase, Control, Plasmid Preparation, Generated, CRISPR, Phospho-proteomics

Journal: Cellular & molecular immunology

Article Title: The zinc-finger protein ZFYVE1 modulates TLR3-mediated signaling by facilitating TLR3 ligand binding.

doi: 10.1038/s41423-019-0265-6

Figure Lengend Snippet: Fig. 3 Zfyve1 deficiency attenuates TLR3-mediated signaling in primary mouse cells. a Effects of Zfyve1 deficiency on the poly(I:C)- and LPS- induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−MLFs (2 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before qPCR analysis. b Effects of Zfyve1 deficiency on the poly(I:C)- and LPS-induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−BMDCs (2 × 105) were or were not treated with poly(I:C) (20 μg/mL) or LPS (100 ng/mL) for the indicated times before qPCR analysis. c Effects of Zfyve1 deficiency on the PGN- or R848-induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−MLFs or BMDCs (2 × 105) were or were not treated with PGN (20 mg/mL) or R848 (10 mM) for 3 h before qPCR analysis. d Effects of Zfyve1 deficiency on the poly(I:C)-induced phosphorylation of IRF3 and p65. Zfyve1+/+ and Zfyve1−/−BMDCs (2 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before immunoblotting analysis. e Effects of Zfyve1 deficiency on the transcription of downstream genes induced by different doses of poly(I:C). Zfyve1+/+ and Zfyve1−/−MLFs (2 × 105) were or were not treated with increasing amounts of poly(I:C) for 3 h before qPCR analysis. The percentage reduction in the transcription of the Ifnb1 and Isg56 genes in Zfyve1−/−MLFs compared with that in Zfyve1+/+ MLFs is shown in the right panels. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test)

Article Snippet: Reagents, antibodies, and cells The following reagents, antibodies, and cells were purchased from the indicated companies: TRIzol (TaKaRa Bio); SYBR Green (BioRad); a dual-specific luciferase assay kit (Promega); polybrene (Millipore); poly(I:C), PGN, and R848 (Invivogen); LPS and DNase I (Sigma); EZ-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), a first strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), a cell mitochondria isolation kit (Beyotime), a nuclear and cytoplasmic Cellular & Molecular Immunology _#####################_ extraction kit (Applygen); ELISA kits to detect murine IFN-β, TNFα, and IL-6 (BioLegend); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (Origene), p-IRF3 (Cell Signaling Technology), p-p65 (Cell Signaling Technology), TLR3 (Cell Signaling Technology), p-Tyr (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), AIF (Santa Cruz Biotechnology), KDEL (Santa Cruz Biotechnology), p65 (Abcam), TBK1 (Abcam), p-TBK1 (Abcam), TRIF (Abcam), LMNB1 (Proteintech), β-tubulin (Life Technology), ZFYVE1 and RAB5A (Abclonal); and Alexa Fluor 488- and Alexa Fluor 594-conjugated goat anti-mouse IgG antibodies (Invitrogen).

Techniques: Phospho-proteomics, Western Blot