Journal: Virology

Article Title: Reactive oxygen species activate NFκB (p65) and p53 and induce apoptosis in RVFV infected liver cells.

doi: 10.1016/j.virol.2013.11.023

Figure Lengend Snippet: Fig. 1. Increased ROS in RVFV infected HepG2 cells. (A) HepG2 cells were infected with the MP-12 strain of RVFV (MOI: 3) and stained after 3 h using MitoSox stain and DAPI. The stained cells were visualized by confocal microscopy. (B) HepG2 cells infected with MP-12 (MOI: 3) were lysed at 24 h post infection and total protein lysate obtained. Lysates from uninfected cells were obtained as controls and both lysates were resolved by SDS-PAGE and western blots, carried out using anti-RVFV and anti-β-actin antibodies.

Article Snippet: Primary antibodies to RVFV (ProSci, Cat# 4519), total p65 (Santa Cruz Biotechnology, Inc. Cat# sc7151), phospho-p65 (ser536) (Santacruz biotechnology, Inc., Cat# 33020), total p53 (Cell Signaling, Cat# 9282S), phospho-p53 (Ser15) (Cell Signaling, Cat# 9284S), Cytochrome C (Abcam, Cat# ab13575) and HRP conjugated actin (Abcam, Cat# ab49900) were used according to manufacturer’s instructions and the blots were incubated overnight at 4 1C.

Techniques: Infection, Staining, Confocal Microscopy, SDS Page, Western Blot

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 1. Blocking of IFN induction by NS1 proteins from several avian influenza A viruses. A549 cells were co-transfected with the pISRE signalling plasmid and NS1 expression plasmid of different influenza viruses for 24 h. Luciferase activity was measured 24 h post-stimulation with poly I : C. The data show the relative luciferase units (RLUs)±SD of three independent experiments performed in duplicate. Allele A NS1s from different isolates showed significantly higher inhibition of ISRE promoter compared with allele B NS1s. *P,0.05 (Student’s t-test). NS1 proteins used for further analysis are represented with white bars, and NS1 proteins from other viruses are shown with grey bars.

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Blocking Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Inhibition

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 2. Alignment of the amino acid sequence for the isolates used in this study. Left panel: a total of 71 (~30 %) amino acids differ in both the RBD and ED. Box indicates the point of mutation while the NLS1 (aa 34–38), NES+regulatory sequence (aa 134–161) and NLS2 (aa 216–230) are underlined. Up headed arrow indicates the NS1 chimeras’s break points. Right panel: the structures of both alleles A and B of H6N8 were predicted using I-TASSER based on multiple-threading alignments, as described previously (Roy et al., 2010) and aligned in MacPymole. The amino acids having similar properties were assigned the same colour.

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Sequencing, Mutagenesis

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 3. Alleles A and B NS1 proteins of avian influenza viruses differentially block the IFN-induced expression of ISRE promoter in A549 and MiLu cells. (a) A549 cells were co-transfected with the pISRE signalling plasmid and the NS1 expression plasmid for 24 h. Luciferase activity was measured 24 h post-stimulation with (dsRNA+) or without (dsRNA”) poly I : C. (b) A549 cells were transfected with the pISRE signalling plasmid for 24 h, followed by infection with corresponding influenza viruses at an m.o.i. of 5 for 10 h and luciferase activity was measured in cell extracts. (c) A similar experimental layout was performed in MiLu cells as that of (a). (d) The same experimental conditions were maintained for MiLu cells as that of (b). Results from separate experiments were combined by setting the RLUs induced by dsRNA in cells transfected with only signalling plasmids to 100 %. The data shown here are the means±SD of three independent experiments performed in duplicate. *, Indicates a significant difference as determined by the Student’s t-test, with P-values of ,0.05, all other pairwise comparisons to control samples were not significant, with P-value of .0.4.

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Blocking Assay, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Infection, Control

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 5. Expression and accumulation patterns of alleles A and B NS1 proteins in subcellular compartments in A549 cells. A549 cells were either lysed or fixed at 18 h post-NS1 trans- fection and subjected to (a) Western blotting or (b) in situ PLA. (c) Quantification of cytoplasmic and nuclear signals was per- formed using BlobFinder software and expressed data represent the means±SD of three microscopic fields.

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Expressing, Western Blot, In Situ, Software

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 6. Subcellular localization pattern of alleles A and B NS1 proteins of H6N8 and H4N6 along with PR8 in A549 and MiLu. A549 and MiLu cells were transfected with indicated NS1 constructs, fixed and stained at 4 and 6 h post-transfection with NS1 poly- clonal sera and visualized using in situ PLA, as recommended.

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Transfection, Construct, Staining, In Situ

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 7. Contribution of F103Y and Y103F substitutions of NS1 protein in blocking the induction of ISRE promoters in A549 and MiLu cells. (a) A549 cells were co-transfected with alleles A and B of H6N8, H6N8-A mutated (F103Y) and H6N8-B (Y103F) mutated NS1s with either ISRE or left untreated for 24 h. (b) MiLu cells were co-transfected with alleles A and B of H6N8, H6N8-A mutated (F103Y) and H6N8-B (Y103F) mutated NS1s with either ISRE or left untreated for 24 h. In all the experiments here, cell lysates were assayed for luciferase activity at 24 h post- poly I : C stimulation. Data are expressed as the means±SD for the three independent experiments performed in duplicate. NS, Represents the non-significant differences at P.0.05 (Student’s t-test).

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Blocking Assay, Transfection, Luciferase, Activity Assay

Journal: The Journal of general virology

Article Title: Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

doi: 10.1099/vir.0.031716-0

Figure Lengend Snippet: Fig. 8. Characterization of the NS1 protein domains in blocking the induction of ISRE promoter in A549 and MiLu cells. (a) A schematic illustration for the construction of chimeric NS1 and other individual domains. (b) Co-transfection of the ISRE signalling plasmid with alleles A and B, RNA and EDs of both alleles A and B of H6N8 and chimeric NS1 of H6N8 as demonstrated in (a). (c) MiLu cells were co-transfected with ISRE plasmid and alleles A and B, RNA and EDs of both alleles A and B of H6N8 and chimeric NS1 of H6N8. In all the experiments here, cell lysates were assayed for luciferase activity at 24 h post- poly I : C stimulation. Data are expressed as the means±SD for the three independent experi- ments performed in duplicate. *, Indicate significant differences at P,0.05 (Student’s t-test).

Article Snippet: After fixation and permeabilization, cells were mixed with primary antibodies raised against the C terminus of NS1 protein [anti-NS1 (ProSci)].

Techniques: Blocking Assay, Cotransfection, Plasmid Preparation, Transfection, Luciferase, Activity Assay