Journal: PLoS ONE

Article Title: Mitochondrial alarmins are tissue mediators of ventilator-induced lung injury and ARDS

doi: 10.1371/journal.pone.0225468

Figure Lengend Snippet: Supernatant collected from 24 hours stretched A549 cells and from unstretched cells were co-incubated with HEK-Blue hTLR9 reported cell line either alone or with TLR9 antagonists ODN TTAGGG. After 24 hours supernatants were analyzed for activity by spectroscopy of the target transgene NF-κB induced secreted embryonic alkaline phosphatase absorbance at 655 nm. Data are expressed as fold change over NST and represent median with interquartile range of at least three independent experiments. * P < 0.05 , Mann-Whitney test (NST vs ST) and Wilcoxon signed-rank test (ST vs ST+ ODN TTAGGG) with FDR correction. NST: non stretch, ST: stretch.

Article Snippet: In some experiments, 2 μM of the TLR9 antagonist ODN TTAGGG (Invivogen, San Diego, CA, USA) was added to the A549 cell cultures just before starting cell stretch.

Techniques: Incubation, Activity Assay, Spectroscopy, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Mitochondrial alarmins are tissue mediators of ventilator-induced lung injury and ARDS

doi: 10.1371/journal.pone.0225468

Figure Lengend Snippet: ( A ) Il-8 concentration measured in supernatant collected from A549 cells submitted to cyclic mechanical stretch for 6, 24 and 48 hours and their control unstretched cells. ( B ) Il-8 concentration measured in supernatant collected from A549 cells stretched for 24 hours and co-incubated with TLR9 antagonist ODN TTAGGG. Data are expressed as fold change over NST and represent median with interquartile range of at least three independent experiments. **P < 0.01 , ***P < 0.001, Mann-Whitney test ( A ), Kruskall-Wallis test with FDR correction ( B ). NST: non stretch, ST: stretch.

Article Snippet: In some experiments, 2 μM of the TLR9 antagonist ODN TTAGGG (Invivogen, San Diego, CA, USA) was added to the A549 cell cultures just before starting cell stretch.

Techniques: Concentration Assay, Incubation, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Mitochondrial alarmins are tissue mediators of ventilator-induced lung injury and ARDS

doi: 10.1371/journal.pone.0225468

Figure Lengend Snippet: Boyden chamber chemotaxis assay of human primary neutrophils stimulated with ( A ) conditioned supernatants collected from A549 cells submitted mechanical stretch for 24 hours and unstretched A549 cells and ( B ) antibodies against the fMLP receptor (anti-FPR1) and the IL-8 receptor (anti-CXCR1). Data are expressed as percentage of NST ( A ) and ST ( B ) and represent median with interquartile range of at least three independent experiments. * P < 0.05 , **P < 0.01 , Mann-Whitney test ( A ), Kruskall-Wallis test with FDR correction ( B ). NST: non stretch, ST: stretch, FPR1: anti-formyl peptide receptor 1, CXCR1: C-X-C motif chemokine receptor 1.

Article Snippet: In some experiments, 2 μM of the TLR9 antagonist ODN TTAGGG (Invivogen, San Diego, CA, USA) was added to the A549 cell cultures just before starting cell stretch.

Techniques: Chemotaxis Assay, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

doi: 10.1128/JVI.02274-20

Figure Lengend Snippet: Inhibiting NP expression induces an antiviral host immune response. (A) Schematic of the recombinant influenza A virus genome containing the miRNA-targeting cassette downstream of the NP open reading frame (ORF). (B) Schematic of the recombinant Sendai virus genome containing the miRNA-targeting cassette downstream of the N ORF. A GFP ORF is also inserted between the N and P ORFs. (C) Western blot analysis for IAV NP, IFIT1, and actin of whole-cell lysates of A549 cells infected with IAV-NP C or IAV-NP T at an MOI of 5 for 0, 6, 12, or 24 h. (D) Western blot analysis for SeV N, IFIT1, and actin of whole-cell lysates of A549 cells infected with SeV-N C or SeV-N T at an MOI of 5 for 0, 6, 12, or 24 h. (E) Heat map analysis of the log 2 (fold change) expression levels of differentially expressed genes involved in the IFN-I response compared to mock-infected cells after bulk mRNA-seq analysis of IAV-NP C /IAV-NP T (MOI of 5; 9 hpi)- or SeV-N C /SeV-N T (MOI of 5; 24 hpi)-infected A549 cells. (F) Mean percentage of viral reads over total mapped reads. (G) Dot plot visualization of enriched GO terms after RNA-seq analysis. Gene set enrichment analysis (GSEA) was performed against the GO data sets for biological processes. The color of the dots represents the false discovery rate (FDR) value for each enriched GO term. The size of the dots represents the enrichment signal strength (as a percentage) of genes included in the complete gene set. Light gray dots represent nonsignificant enrichments (FDR ≥ 0.05).

Article Snippet: A549-Dual, A549-Dual-KO-MAVS, A549-Dual-KO-MDA5, and A549-Dual-KO-RIG-I cells were acquired from InvivoGen and maintained in DMEM supplemented with 10% FBS, 100 μg/ml Normocin, 10 μg/ml blasticidin, and 100 μg/ml zeocin.

Techniques: Expressing, Recombinant, Virus, Western Blot, Infection, RNA Sequencing Assay

Journal: Journal of Virology

Article Title: Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

doi: 10.1128/JVI.02274-20

Figure Lengend Snippet: Limiting NP expression induces the production of defective viral genomes. (A) Read coverage along the IAV-NP C (black) or IAV-NP T (magenta) viral genomes in A549 cells infected at an MOI of 5 for 9 h after bulk mRNA-seq. (B) Mean percentage of viral reads over total mapped reads of total rRNA-depleted RNA-seq of IAV-NP C - or IAV-NP T -infected A549 cells (MOI of 5; 9 hpi). (C) Proportion of viral reads corresponding to panel A that map to the 5′ (15%)- and 3′ (15%)-terminal regions of each IAV segment. (D) Proportion of viral reads corresponding to panel B that map to noncanonical junction reads. (E) Read coverage along the SeV-N C (black) or SeV-N T (magenta) antigenome in A549 cells infected at an MOI of 5 for 9 h after ribosome-depleted total RNA-seq. (F) Mean percentage of viral reads over total mapped reads. (G) Proportion of viral reads that map to the 3′-terminal (15%) region of the SeV anti-genome. Data are derived from two independent biological replicates, with error bars depicting the standard deviation. Statistical significance was determined by an unpaired two-sample two-tailed t test. *, P < 0.05; **, P < 0.01.

Article Snippet: A549-Dual, A549-Dual-KO-MAVS, A549-Dual-KO-MDA5, and A549-Dual-KO-RIG-I cells were acquired from InvivoGen and maintained in DMEM supplemented with 10% FBS, 100 μg/ml Normocin, 10 μg/ml blasticidin, and 100 μg/ml zeocin.

Techniques: Expressing, Infection, RNA Sequencing Assay, Derivative Assay, Standard Deviation, Two Tailed Test

Journal: Journal of Virology

Article Title: Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

doi: 10.1128/JVI.02274-20

Figure Lengend Snippet: NP expression regulates the production of defective viral genomes and the induction of the antiviral host response. (A) Northern blot analysis of RNA extracted from A549 cells infected with IAV-NP C or IAV-NP T at an MOI of 5. Radiolabeled probes against the conserved 5′ vRNA promoter of IAV and U6 snRNA (106 nucleotides [nt]) as an internal loading control were used. (B) RT-PCR analysis for the presence of IFN-β and α-tubulin mRNA from samples used for panel A. (C) Northern blot analysis of RNA extracted from HEK-293T cells transiently expressing a truncated 200-nt-long IAV segment 6 vRNA (containing only the 100 terminal nucleotides at the 3′ and 5′ vRNA ends) together with constant amounts of IAV RdRp and increasing amounts of IAV NP. A catalytically inactive RdRp (PB1a) was used as a negative control. Radiolabeled probes against the conserved 5′ vRNA promoter of IAV and U6 snRNA (106 nt) as an internal loading control were used. (D) RT-PCR analysis for the presence of IFN-β and α-tubulin mRNA from samples used for panel C. (E) Luciferase reporter assay for IFN expression in wild-type (WT), ΔMAVS, ΔRIG-I, or ΔMDA5 A549-Dual cells infected with IAV-NP C or IAV-NP T at an MOI of 5 for 12 h. The graph shows the mean fold change of relative light units (RLU) compared to mock-infected cells from three independent biological replicates, with error bars representing the standard deviation. Statistical significance was determined by unpaired two-sample two-tailed t test. ns, not significant ( P > 0.05); **, P < 0.01; ***, P < 0.001. (F) Western blot analysis of whole-cell lysates of A549 cells infected with IAV at an MOI of 5 for 8 h in the presence of increasing concentrations of Nz (0.1, 0.2, 0.3, and 0.4 μM) or BXM (1, 10, 25, and 50 μM).

Article Snippet: A549-Dual, A549-Dual-KO-MAVS, A549-Dual-KO-MDA5, and A549-Dual-KO-RIG-I cells were acquired from InvivoGen and maintained in DMEM supplemented with 10% FBS, 100 μg/ml Normocin, 10 μg/ml blasticidin, and 100 μg/ml zeocin.

Techniques: Expressing, Northern Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Luciferase, Reporter Assay, Standard Deviation, Two Tailed Test, Western Blot

Journal: Journal of Virology

Article Title: Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition

doi: 10.1128/JVI.02274-20

Figure Lengend Snippet: Limiting NP expression induces a strong antiviral response for NSVs. (A) Flow cytometry analysis of A549 cells transfected with nontargeting or NP-targeting siRNA pools prior to infection with the indicated viruses. The graph shows the mean percentage of fluorescent-positive cells from three independent biological replicates, with error bars representing the standard deviation. (B) Flow cytometry analysis of A549 cells transfected with nontargeting or NP-targeting siRNA pools prior to infection with the indicated viruses. The graph shows the mean fluorescent intensity of each cell from three independent biological replicates, with error bars representing the standard deviation. Significance was determined by two-sample two-tailed t tests. ns, not significant ( P > 0.05); ***, P < 0.001; ****, P < 0.0001. (C to I) A549 cells were transfected with nontargeting or NP-targeting siRNA pools prior to infection with the indicated viruses. Whole-cell lysates were analyzed by Western blotting for IFIT1, actin, and viral protein for (C) IAV-mNeon, (D) LASV-tdTom, (E) EBOV-GFP, (F) VSV-GFP, (G) MeV-GFP, (H) HPIV3-GFP, and (I) RSV-GFP. (J) A549 ACE2 cells were transfected with nontargeting or subgenomic N-targeting siRNA prior to infection with SARS-CoV-2. Whole-cell lysates were analyzed by Western blotting for IFIT1, actin, and SARS-CoV-2 nucleocapsid. A549 ACE2 cells transfected with 1 μg poly(I·C) instead of siRNA were used as a positive control. (K) A549 ACE2 cells were transfected with nontargeting or subgenomic N-targeting siRNA prior to infection with SARS-CoV-2 at an MOI of 0.1 for 24 h. The graph shows the mean percentage of SARS-CoV-2 reads over total mapped reads from bulk mRNA-seq from three independent biological replicates, with error bars representing the standard deviation. Significance was determined by two-sample two-tailed t tests. ****, P < 0.0001.

Article Snippet: A549-Dual, A549-Dual-KO-MAVS, A549-Dual-KO-MDA5, and A549-Dual-KO-RIG-I cells were acquired from InvivoGen and maintained in DMEM supplemented with 10% FBS, 100 μg/ml Normocin, 10 μg/ml blasticidin, and 100 μg/ml zeocin.

Techniques: Expressing, Flow Cytometry, Transfection, Infection, Standard Deviation, Two Tailed Test, Western Blot, Positive Control