Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. Schematic diagram of the engineered F-TrCP-Shc ubiquitin ligase to target the ErbB RTKs. B. F-TrCP-Shc binds to ErbB2. 293T cells were transiently transfected with the indicated plasmids. ErbB2 was immunoprecipitated with the anti-ErbB2 antibody and immunoblotted with the ErbB2, FLAG and β-actin antibodies. C. Degradation of endogenous ErbB2 by the engineered F-TrCP-Shc. 293T cells were transiently transfected with increasing doses of F-TrCP-Shc or F-TrCP-Shc(m), and levels of endogenous ErbB2, F-TrCP fusions and β-actin were determined by Western blotting. The experiment was repeated three times. D. F-TrCP-Shc promotes ubiquitination of ErbB2. 293T cells were transfected with the indicated plasmids for in vivo ubiquitination assays. E–F. F-TrCP-Shc-induced ErbB2 degradation is proteasome-dependent. SKBR3 and BT474 cells expressing F-TrCP-Shc, F-TrCP-Shc(m) or pBMN-GFP were treated with 50 μM MG132 or 100 μM choloroquine. The levels of endogenous ErbB2, F-TrCP fusions and β-actin were determined by Western blotting.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, In Vivo, Expressing

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: SKBR3 (A) and BT474 (B) cells infected with the indicated recombinant retroviruses were lysed and 50 μg of each cell lysate was analyzed by SDS-PAGE and Western blotting with the indicated antibodies. C. SKBR3 and BT474 cells were plated in 6-well plates for 24 hours, trypsinized and washed in FACS buffer. The live cells were stained with anti-ErbB2 antibody (Santa Cruz) against the extracellular domain of ErbB2. Results are the mean for three replicate experiments. D. Dose-dependent degradation of endogenous ErbB2 upon infection of adenoviral F-TrCP-Shc was determined by Western blotting with the indicated antibodies. E. Depletion of ErbB2 on the cell membrane by adenoviral F-TrCP-Shc. ErbB2 (red) was detected by immunofluorescent staining with the anti-ErbB2 antibody (R&D Systems). Infected cells were marked by GFP expression from the adenoviral vector. The nuclei were visualized by DAPI staining (blue). White arrow, a low F-TrCP-Shc expressing SKBR3 cell.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, Recombinant, SDS Page, Western Blot, Staining, Membrane, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. BT474 cells were infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP, sorted and analyzed for inhibition of MAPK and PI3K signaling by Western blotting as in Fig. 4A. B. Retroviral expression of F-TrCP-Shc in BT474 cells inhibited cell growth. Results are the mean for three replicate experiments. * indicates P<0.05 for pBMN-F-TrCP-Shc-infected cells compared to those with pBMN-GFP or pBMN-F-TrCP-Shc(m). C–D. Apoptotic induction of BT474 cells by F-TrCP-Shc, as determined by Annexin V-FACS analysis and PARP1 cleavage by Western blotting. For FACS analysis, cells were stained with Annexin-V-APC and 7-AAD, and the percentage of early ( right bottom ) and late apoptotic ( right top ) populations are indicated.

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, Inhibition, Western Blot, Retroviral, Expressing, Staining

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: The engineered F-TrCP-Shc E3 ligase sensitizes BT474 breast cancer cells to cytotoxic killing by cisplatin and decreases their transforming ability. A–B. BT474 or MCF-10A cells infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP retroviruses were treated with the indicated amounts of cisplatin for 48 hours, and the cell viability was measured by the XTT assay. Results are represented as the mean ± standard deviation (n=3), for BT474 cells. C–D. Focus formation assay. Retroviral vector infected BT474 cells were seeded in 6-well plates and cultured for 10 days to allow for colony formation. The colonies were fixed, stained and photographed. The colony numbers were counted and the colony formation ratio was calculated according to the formula: colony formation ratio ( % ) = ( number of colonies / number of cells seeded ) × 100 . Results are represented as the mean ± standard deviation (n=3). * indicates P<0.05 for pBMN-F-TrCP-Shc-infected cells compared to those with pBMN-GFP or pBMN-F-TrCP-Shc(m).

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Infection, XTT Assay, Standard Deviation, Tube Formation Assay, Retroviral, Plasmid Preparation, Cell Culture, Staining

Journal: Oncogene

Article Title: Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases

doi: 10.1038/onc.2013.33

Figure Lengend Snippet: A. Nude mice were injected subcutaneously in the right flank with 5×10 6 BT474 cells infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc(m) or pBMN-GFP retroviruses. Tumor volumes were measured over a 4-week period and calculated by the formula: ( length × width 2 ) / 2 . B. Tumor weight (g) was recorded at the end of the experiment. Columns, mean value of tumor weight; bars, SD. C. Representative images of tumor-bearing mice. * P<0.05 or **P<0.01 indicate tumors expressing pBMN-F-TrCP-Shc compared to those infected with pBMN-GFP or pBMN-F-TrCP-Shc(m).

Article Snippet: Human mammary carcinoma cell lines SKBR3 and BT474, and non-cancerous MCF-10A mammary epithelial cells were obtained from ATCC.

Techniques: Injection, Infection, Expressing

Journal: The Journal of infectious diseases

Article Title: Immunomodulatory Function of Interleukin 28B during primary infection with cytomegalovirus.

doi: 10.1093/infdis/jiu144

Figure Lengend Snippet: Figure 2. IL-28B SNP in fibroblasts reduces CMV replication by increased CMV-induced IFN-α and reduced IFN-λ responses. HFF cells with different IL-28B genotypes (HS97FS rs12979860: CC, rs8099917: TT; SCRC1041 rs12979860: CT, rs8099917: TT; and CCD1112SK rs12979860: TT, rs8099917: TT) were infected with human CMV (Towne strain, MOI 0.3). mRNA expression is relative to HPRT and non-infected controls, and 3 independent experiments were performed unless otherwise indicated. Bars or symbols indicate median value, whiskers the interquartile ranges unless otherwise indicated. The rs8099917 was the same for all tested cell lines (major allele genotype). A, CMV replication in HFF cells measured with plaque assays. Supernatants were collected at day 4 from 9 individual experiments. Viral growth was determined using plaque assays as described, and viral load is expressed as plaque- forming units (PFU) per mL of supernatant. B, IFN-λ mRNA-expression of infected fibroblasts. IL-28A, IL-28B, and IL-29 mRNA is expressed at 24 hours. C, IFN-α2 mRNA expression of stimulated or infected fibroblasts. In addition to infection (as previously described), a stimulation with poly I:C (50 μg/mL) was performed. mRNA expression of IFN-α2 is shown at 12 hours. D, Proinflammatory ISG response (IFIT2 and OAS1) during CMV infection of HFF cells. E, Proinflammatory ISG response (Mx1 an ISG15) during CMV infection of HFF cells. F, Anti-inflammatory ISG response (SOCS1 and USP18) during infection and stimulation of HFF cells. Same infection condition as previously describe were used, but in this experiment stimulation was performed with transfected poly I:C (7.5 μg/mL). Abbreviations: CMV, cytomegalovirus; HFF, human foreskin fibroblast; IFN, interferon; IL, interleukin; ISG, IFN-stimulated gene; mRNA, messenger RNA.

Article Snippet: The following primers and probes were used (TaqMan gene expression assays): Interferon alpha-2 (IFNα2, Hs00265051_s1), Interferon beta-1 (IFNβ1, Hs01077958_s1), MX1 (Hs00895608_m1), OAS1 (Hs00973637_m1), IFIT2 (Hs 00533665_m1), ISG15 (Hs01921425_s1), SOCS1 (00705164_ s1), USP18 (Hs00276441_m1), HPRT (Hs01003267_m1), and IL-28RA (Hs00417120_m1).

Techniques: Infection, Expressing, Transfection

Journal: The Journal of infectious diseases

Article Title: Immunomodulatory Function of Interleukin 28B during primary infection with cytomegalovirus.

doi: 10.1093/infdis/jiu144

Figure Lengend Snippet: Figure 5. Modulating effects of IL-28B and IL-28B SNPs on interferon responses and on adaptive immune functions during T-cell priming. PBMCs from healthy volunteers were stimulated with CMV (MOI 0.3). IFN-λ and ISG mRNA-expressions was determined A and B, mRNA was normalized to HPRT expression and relative fold increases were calculated to non-stimulated controls. Bars indicate median values, whiskers interquartile range values. Com- parison between 4 major-allele carriers and 4 minor-allele carriers are shown. A, CMV induced IFN-λ mRNA expression according to IL-28B genotype. mRNA-expression is shown 6 hours after stimulation. B, Stimulatory effect of CMV on pro-inflammatory ISG mRNA expression according to genotype. IFNα2 mRNA-expression is shown 6 hours later, and MX1 and OAS1 mRNA expression are shown at 72 hours time point. C, Effect of IL-28B on pro-inflammatory ISG expression. PBMCs were pretreated for 2 hours with recombinant IL-28B (100 ng/mL) and then stimulated with CMV (MOI 0.3). MX1, OAS1, IFIT2, ISG15 mRNA-expression is shown at 72 hours irrespective of the IL-28B genotype. Comparisons between 7 individuals are shown. D, Effects of pretreatment of PBMC with peptides on CMV stimulated IFN-α production. Irrespective of the IL28B genotype, PBMC from healthy volunteers (n = 5) were pretreated with peptides (10 μM) for 2 hours prior to stimulation with CMV (MOI 0.3). IFNα levels were determined by ELISA at 24 hours. IFNα is shown as fold change compared to non-peptide treated controls. E, Effects of pre-treatment of PBMC with peptides on CMV stimulated ISG mRNA expression. Relative ISG expressions of MX1, OAS1, and IFIT2 were pooled in a profile for every single treatment condition (a separation is shown in Supplementary Figure 2A). F, Impact of IL-28B on antigen presentation and co-stimulatory signaling of monocytes. PBMCs from CMV seronegative healthy blood donors were pre- treated with recombinant IL-28B (100 ng/mL) prior to a 5-day stimulation with CMV (MOI 0.05). Relative MFI expression of CD86, HLA DR, CD40 and CD14 is shown normalized to noninfected and non-pretreated fibroblast lysate (raw data of the experiment is provided in Supplementary Figure 2B). G, Impact of IL-28B on priming of naive T cells. PBMCs from CMV seronegative healthy volunteers were pretreated with recombinant IL-28B (100 ng/mL) prior to a 5-day stimulation with CMV (MOI 0.3). T-cell proliferation during in vitro priming with CMV is expressed in percent of the overall respective CD4 T-cell population. H, Impact of IL-28B on T-cell activation after expansion phase. T cells of different subtypes show a clear down-regulation of CD69 surface expression. Abbreviations: CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; HFF, human foreskin fibroblast; IFN, interferon; IL, interleukin; ISG, IFN-stimulated gene; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cells; siRNA, small interfering RNA.

Article Snippet: The following primers and probes were used (TaqMan gene expression assays): Interferon alpha-2 (IFNα2, Hs00265051_s1), Interferon beta-1 (IFNβ1, Hs01077958_s1), MX1 (Hs00895608_m1), OAS1 (Hs00973637_m1), IFIT2 (Hs 00533665_m1), ISG15 (Hs01921425_s1), SOCS1 (00705164_ s1), USP18 (Hs00276441_m1), HPRT (Hs01003267_m1), and IL-28RA (Hs00417120_m1).

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Immunopeptidomics, In Vitro, Activation Assay, Small Interfering RNA

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Different cell types composing intestinal wall were examined for the presence of Fc gamma receptors. Villin and α-SMA were used to confirm cell type-specific phenotypes (A). Immunofluorescence analysis of CD64-expressing CCD-18Co fibroblasts. Cells expressing CD64 at the plasma membrane are indicated by arrows. Size bar: 50 µm (B). Intestinal fibroblasts CCD-18Co (C), intestinal epithelial cells Caco2 BBE (D) and THP-1 cells (E) were pre-incubated with anti-TNF therapeutics, and subsequently treated with human recombinant TNF to induce pro-inflammatory response. Values on Y axis represent mRNA expression levels relative to ß-actin. Columns represent mean values of four independent experiments measured in triplicate. Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Immunofluorescence, Expressing, Clinical Proteomics, Membrane, Incubation, Recombinant

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Peripheral blood of healthy donors (n = 4) and IBD patients (n = 3) was treated with different anti-TNFs and subsequently with soluble TNF to induce pro-inflammatory responses in PBMCs and plasma. The mRNA expression levels of CD64 (A), CD32 (B) and CD16 (C) Fc gamma receptors in healthy individuals and IBD patients were assessed by RT-PCR as compared to THP-1 cells (set to 1). The extent of inhibition by each drug was determined by measuring mRNA levels of ICAM-1 (D), TNF (E), and IL-8 (F) after 2 h of TNF stimulation, as well as by the release of IL-8 after 24 h of combined TNF and anti-TNF stimulation (G). The inhibitory efficacies were determined after eliminating intra-individual differences by setting the TNF-induced responses to 100% for each donor and target (H–K). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Clinical Proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Both ADA and IFX immune complexes were added to THP-1 cells to activate CD64 receptor in the presence (+FCS) or absence (−FCS) of serum. ADA/TNF complexes were more potent in inducing phospho-tyrosine signaling (A), but IFX either alone or in complex with TNF induced phosphorylation of distinct tyrosine residues (enlarged insert, right panel). Activation of CD64 downstream signaling leads to elevated transcription of GM-CSF (B, C), MCP-1 (D, E) and IL-8 (F, G) genes. Human serum (HS) was used as a positive control for activation of Fc gamma receptor(s). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Phospho-proteomics, Activation Assay, Positive Control

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: CCD-18Co fibroblasts (A), Ko77 fibroblasts (C) and THP-1 cells (E) were pre-incubated with human IgG1 isotype (10 µg/ml) prior to subsequent incubation with infliximab (1 µg/ml) and TNF (50 ng/ml). Columns show mean values from a single, representative experiment measured in triplicates. Error bars indicate SD of three measurements. The recovery of anti-TNF activity was also evaluated as percentage of inhibition of TNF-induced responses for each cell line (B, D, F).

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Incubation, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Blocking of Fc fragments of adalimumab and infliximab changes their inhibitory efficacy in Ko77 fibroblasts (A). Graph shows mean values from four independent experiments measured in triplicates. Error bars represent SD. Fab fragments of anti-TNFs have different inhibitory efficacies as compared to the whole IgG1 molecules in THP-1 cells (B). Columns represent mean values of three independent experiments measured in triplicates and set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: Treatment with IFN-γ elevates the mRNA expression levels of CD64 (A), which result in the loss of efficacy of IFX and ADA (B). Decreased expression levels of CD64 (C) correlate with increased inhibitory efficacy of IFX (D). PMA treatment decreases the expression levels of CD64 (E), which results in complete recovery of efficacy of IFX (F). Graphs show mean values of at least three independent experiments measured in triplicates and columns are set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab

doi: 10.1371/journal.pone.0043361

Figure Lengend Snippet: IFN-γ induces its receptor downstream signaling involving JAK and STAT proteins (A). STAT dimers elevate the transcription of the CD64 (FCGR1) gene (B). Translated CD64 protein is targeted into plasma membrane, where it resides in an inactive state until binding immune complexes IFX-TNF (C). Propagation of downstream signaling (D) occurs through immunoreceptor tyrosine-based activation motif (ITAM) leading to the transcription of relevant target genes (E) via parallel activation of p38, JNK and ERK kinases . Production and release of IL-8 and other pro-inflammatory factors contribute to the limited outcome of IFX treatment.

Article Snippet: CCL2 (Hs00234240_m1), CSF2 (Hs00929873_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), ICAM-1 (Hs00164932_m1), CD68 (Hs00154355_m1), FCGR1A (Hs00174081_m1), FCGR2A (Hs01017702_g1), FCGR3 (Hs00275547_m1), TNF (Hs00174128_m1) and β-actin (4310881E) human gene expression assays were provided by Applied Biosystems (Foster City, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Activation Assay

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