virus infection human lung epithelial cell line a549  (ATCC)

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 overexpression inhibits influenza virus PR8 replication and induces RIG-I and IFN-β expression in A549 cells. A549 cells were transfected with 2 μg of vector alone, myc-NLRC5 or flag-CIITA expression vector. Twenty-four hours post-transfection, the cells were infected with PR8 virus (MOI 0, 0.01, 0.1, and 1.0) for 24 h. (A) Supernatants were tested for viral titers by plaque assay using MDCK cells. (B–F) The expression of (B) NP vRNA, (C) NP mRNA, (D) IFN-β mRNA, (E) RIG-I mRNA, and (F) IFN-α was analyzed by real-time RT-PCR, relative to β-actin. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. (F) Expression of myc-NLRC5 and NS1 was analyzed by immunoblotting and the immunoblot shown is from one single experiment representative of three independent experiments. β-Actin was used as a loading control. ANOVA was performed to compare vector control versus myc-NLRC5 or flag-CIITA-transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Over Expression, Virus, Expressing, Transfection, Plasmid Preparation, Infection, Plaque Assay, Quantitative RT-PCR, Western Blot, Control

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: Viral NS1 counteracts endogenous NLRC5, RIG-I, and IFN-β expression. A549 cells were infected with PR8 or PR8ΔNS1 (MOI 1.0) for 0, 3, 6, 12, and 24 h and the expression of (A) NP vRNA, (B) NP mRNA, (C) NLRC5 mRNA, (D) RIG-I mRNA, (E) IFN-β mRNA, and (F) IFN-α was analyzed by real-time RT-PCR, relative to β-actin. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare PR8-infected versus PR8ΔNS1-infected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NS1 complementation inhibits PR8ΔNS1-induced NLRC5. A549 cells were cotransfected with 2 μg of vector or myc-NS1 expression vector and IFN-β promoter LUC reporter using lipofectamine 2000. Twenty-four hours post-transfection, these cells were infected with PR8ΔNS1 for another 24 h. (A, B) Cells were harvested and mRNA expression of (A) NLRC5 and (B) RIG-I was determined by real-time RT-PCR, relative to β-actin. (C) IFN-β induction was assayed by LUC reporter assay. (D) NLRC5, RIG-I, and myc-NS1 expression was analyzed by immunoblotting. The immunoblot shown is from one single experiment representative of three independent experiments. β-Actin was used as a loading control. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare vector control versus myc-NS1-transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Plasmid Preparation, Expressing, Transfection, Infection, Quantitative RT-PCR, Reporter Assay, Western Blot, Control

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NS1ΔPR8 virus induces NLRC5, IFN-β, RANTES expression, and NFκB activation in a RIG-I-dependent manner. The expression of endogenous NLRC5 or RIG-I was silenced using gene-specific NLRC5 or RIG-I siRNA in A549 cells followed by infection with PR8 or PR8ΔNS1 (MOI 1.0). Cells were also cotransfected with NFκB promoter LUC reporter using lipofectamine 2000. (A–C) Cells were harvested 24 h postinfection to assess the expression of (A) NLRC5 mRNA, (B) IFN-β mRNA, and (C) RIG-I mRNA, relative to β-actin by real-time RT-PCR. (D) Cells were analyzed for endogenous RIG-I, NLRC5, and β-actin (loading control) protein expression by immunoblotting and the immunoblot shown is from one single experiment representative of three independent experiments. Cell supernatants were assayed for (E) IFN-β and (F) RANTES by ELISA. (G) NFκB activation was measured by LUC reporter assay. Data shown are mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare PR8-infected versus PR8ΔNS1-infected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Virus, Expressing, Activation Assay, Infection, Quantitative RT-PCR, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Reporter Assay

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: LPS-induced IFN-β induction and NFκB activation remain unchanged in the presence or absence of NLRC5. (A–C) The expression of endogenous NLRC5 in A549 cells was silenced using gene-specific NLRC5 siRNA. (D–F) Alternatively, A549 cells were transfected with 2 μg of vector alone or myc-NLRC5 expression vector. (A–F) Cells were also cotransfected with IFN-β promoter or NFκB promoter LUC reporter using lipofectamine 2000 and treated with indicated dose of LPS. (A–F) Cells were harvested 24 h post-LPS treatment to assess (A and D) IFN-β induction and (B and E) NFκB activation by LUC reporter assay. (C and F) Cells were analyzed for myc-NLRC5, RIG-I, and β-actin (loading control) protein expression by immunoblotting and the immunoblot shown is from one single experiment representative of two independent experiments. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control siRNA versus NLRC5 siRNA treated A549 cells or vector versus NLRC5-transfected A549 cells and p values <0.05 are indicated with an asterisk; ns: not significant.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Reporter Assay, Control, Western Blot

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 is required for robust IFN-β and RIG-I expression. A549 cells transfected with control siRNA or NLRC5 siRNA were infected with NS1-del PR8 (MOI 1.0). (A–C) Cells were harvested 0, 24, 48, 72, and 96 h postinfection and analyzed for (A) IFN-β, (B) RIG-I, and (C) NLRC5 mRNA expression, relative to β-actin by real-time RT-PCR. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control siRNA versus NLRC5 siRNA treated A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Expressing, Transfection, Control, Infection, Quantitative RT-PCR

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: The NLRC5 death domain and nucleotide-binding domain is critical for NLRC5-mediated antiviral function. A549 cells were transfected with vector alone or with myc-tagged wtNLRC5, NLRC5-K234A, NLRC5-ISO3, NLRC5-ΔDD, NLRC5-DD, or LRR domain of NLRC5 and subsequently infected with PR8 (MOI 1.0) for 24 h. (A) The upper panel shows the schematic representation of the NLRC5 constructs used and the lower panel shows expression of NLRC5 constructs in the cells by immunoblotting. β-Actin was used as a loading control. (B, C) Supernatants were collected and cells were harvested to determine (B) viral titers by plaque assay and (C) IFN-β mRNA expression, relative to β-actin by real-time RT-PCR. (D) Secretion of CCL5 (RANTES) in cell supernatants was measured by ELISA. Data are shown as mean + SD of three samples per group, pooled from three independent experiments carried out in duplicate. ANOVA was performed to compare control vector versus myc-NLRC5 expression vectors transfected A549 cells and p values <0.05 are indicated with an asterisk.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Binding Assay, Transfection, Plasmid Preparation, Infection, Construct, Expressing, Western Blot, Control, Plaque Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection

doi: 10.1002/eji.201344412

Figure Lengend Snippet: NLRC5 stabilizes RIG-I. (A) A549 cells transfected with vector alone or myc-wtNLRC5 were infected with PR8 (MOI 1.0) for 0, 3, 6, 9, 12, 18, 24, 48, and 72 h and harvested for NLRC5, RIG-I, and NS1 expression and coimmunoprecipitation assay. β-Actin was used as a loading control. Cell lysates from 6, 12, and 24 h were immunoprecipitated with anti-myc, anti-NS1, or anti-RIG-I antibodies and immunoprecipitates were analyzed for the presence of RIG-I, NS1, and NLRC5 by immunoblotting. (B) To map the domain responsible for NLRC5 interaction with RIG-I and NS1, A549 cells were transfected for 24 h with myc-vector alone or with myc-tagged wtNLRC5, NLRC5-K234A, NLRC5-ISO3, NLRC5-ΔDD, NLRC5-DD, or NLRC5-LRR mutants and then infected with PR8 (MOI 1.0) for 3 or 9 h. Cell lysates were immunoprecipitated with anti-myc, anti-NS1, or anti-RIG-I antibodies and immunoprecipitates were analyzed for the presence of RIG-I, NS1, and NLRC5 by immunoblotting. β-Actin was used as a loading control. The input for the immunoblot was about 5% of the total cell lysate. (C) A549 cells transfected with wtNLRC5 were infected with PR8 (MOI 1.0) in the presence or absence of actinomycin D (5 μg/mL)/cyclohexamide (20 μg/mL) combination. Cell lysates were analyzed for RIG-I and NLRC5 expression at 0, 3, 6, and 9 h postinfection by immunoblotting. Data shown are from one single experiment representative of two independent experiments.

Article Snippet: Cell cultures and virus infection Human lung epithelial cell line A549, HEK293T (ATCC, VA, USA), and NHBE cells (Lonza, Switzerland) were maintained as described [ 13 , 26 ].

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Co-Immunoprecipitation Assay, Control, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action

doi: 10.1038/ncomms14234

Figure Lengend Snippet: ( a ) A549 cells infected with B/Brisbane/60/2008 were labelled with 46B8, Rituximab (negative control) or Cetuximab (positive control) prior to incubation with NK cells. NK cell activation (left panel) and target cell lysis (right panel) were presented as the frequency of CD107a + cells and the percent of LDH release, respectively. ( b ) A549 cells infected with B/Brisbane/60/2008 were labelled with 46B8 or 46B8 N297G prior to incubation with NK cells. Target cell lysis was presented as the percent of LDH release. The assays were done in duplicate. Results are representative of three independent experiments. ( a , b : mean and s.e.m.)

Article Snippet: For CDC assay, virus-infected A549 cells or WIL2-S cells (ATCC, CRL-8885, expressing CD20) were coated with varying concentrations of 46B8 or Rituximab.

Techniques: Infection, Negative Control, Positive Control, Incubation, Activation Assay, Lysis

Journal: Nature Communications

Article Title: A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action

doi: 10.1038/ncomms14234

Figure Lengend Snippet: ( a ) A549 cells infected with WT or mutant B/Brisbane/60/2008 viruses were labelled with 46B8 prior to incubation with NK cells. NK cell activation (left panel) and target cell lysis (right panel) were presented as the frequency of CD107a + cells and the percent of LDH release, respectively. The assays were done in duplicate. Results are representative of three independent experiments. ( b , c ) DBA/2 J mice were infected intranasally with a minimum lethal dose of the WT or mutant B/Brisbane/60/2008 viruses. At 72 h post infection, mice received a single treatment of 46B8, 46B8 N297G or a control IgG intravenously at 15 mg kg −1 . ( b ) Survival curves. Each group contains eight mice. Log-rank tests of 46B8- versus control IgG-treated groups for all viruses, 46B8 N297G- versus control IgG-treated groups for WT, B1 and C2 and 46B8- versus 46B8 N297G-treated groups for A4, B1 and C2 give P <0.05. ( c ) Percent of average BW of survived mice as compared with the average pre-infection weight. ( a : mean and s.e.m.; c : mean and s.d.)

Article Snippet: For CDC assay, virus-infected A549 cells or WIL2-S cells (ATCC, CRL-8885, expressing CD20) were coated with varying concentrations of 46B8 or Rituximab.

Techniques: Infection, Mutagenesis, Incubation, Activation Assay, Lysis, Control