Journal: PLoS Pathogens

Article Title: IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

doi: 10.1371/journal.ppat.1002337

Figure Lengend Snippet: A) MDCK or (B) A549 cell lines, stably overexpressing IFITM3 or the empty vector alone, were incubated with either the acidophilic dye acridine orange (AO), LTRed, or a flourogenic cathepsin-L substrate (Cath-L). All cells were also stained for DNA (blue). After incubation cells were imaged on a confocal microscope. Middle panels show enlarged images of the IFITM3 cells. (Scale bars: 20 µm throughout). C) Vector (blue) or IFITM3 (red) transduced cell lines, either MDCK (left) or A549 (right), were incubated with LTRed then analyzed by flow cytometry. D) A549 cells stably transduced with IFITM3 or with the vector alone were incubated with LTRed (red), then immunostained for confocal imaging of LC3 (endogenous, green). DNA = blue. E) MDCK cells stably transduced with IFITM3 or with the vector alone were immunostained for confocal imaging of LC3 (endogenous, red) and CD63 (endogenous, green). DNA = blue. F) Confocal images of MDCK cells overexpressing IFITM3 or the empty vector alone showing the distribution and fluorescence intensities of a stably expressed mCherry-EGFP-LC3B fusion protein using fluorescence channels that detect light emitted from the mCherry protein, EGFP or both (merge). DNA = blue. G) Model of IFITM3-mediated restriction of virus replication. Endocytosed viruses enter late endosomes where IFITM3 is present. IFITM3 prevents viral fusion within the endosomes and likely lysosomes via an unknown mechanism, perhaps by altering pH, membrane characteristics, lipid composition, transport speed or destination. Trapped viruses are trafficked to lysosomes and/or autolysosomes where they undergo degradation.

Article Snippet: The following antibodies were used in this study for either Western blotting (WB) or immunoflourescence (IF), or both as indicated, along with their respective source and catalogue number: Primary antibodies: Actin (Sigma A5316, WB), CD63 (Developmental Studies Hybridoma Bank (DSHB) clone H5C6, IF), Fragilis (mouse Ifitm3) (Abcam ab15592, WB, IF), GAPDH (BD Biosciences 610340, WB), HA (Wistar collection, Coriell Institute, clone H18-S210, WC00029, IF), IFITM3 (Abgent AP1153a, WB, IF), IFITM3 (Abgent AP1153c, IF), LAMP1 ((DSHB) clone H4A3, WB, IF), LC3 (Nanotools Mab LC3-5F10, WB, IF), MX1 (Proteintech 13750-1-AP, WB, IF), NP (Millipore clone H16-L10-4R5 MAB8800, IF), RAB7 (Abcam 50533, WB, IF).

Techniques: Stable Transfection, Plasmid Preparation, Incubation, Staining, Microscopy, Flow Cytometry, Transduction, Imaging, Fluorescence

Journal: Journal of Cell Science

Article Title: Evidence for a regulated Ca 2+ entry in proximal tubular cells and its implication in calcium stone formation

doi: 10.1242/jcs.225268

Figure Lengend Snippet: Expression, localization and functionality of transcellular pathway machinery in mouse kidney. (A) Transcript levels in WT whole kidney tissue; WT PT cells and TRPC3 KO PT cells were analyzed by RT-PCR and representative gel images were obtained. Gene expression was quantified, normalized to Gapdh and represented as a bar graph. (B,C) Distribution and localization of (B) NCX1 (red; highlighted with white arrows) and (C) PMCA1 (green; highlighted with purple arrows) were determined by immunofluorescence staining using megalin (green; highlighted with yellow arrows) as a PT marker in WT mouse kidney sections. (D) Ca2+ imaging traces of WT PT cells showing L-Phe-induced Ca2+-flux and the effect of PMCA inhibitor (carboxyeosin; 10 µM) and NCX inhibitor (KB-R7943; 15 µM). Corresponding (E) Ca2+ peak entry, (F) Ca2+ entry rise and (G) Ca2+ entry reduction are represented as bar diagrams. Results represent means±s.e.m. from n=4 experiments. **P<0.01 (unpaired two-tailed t-test for A and E–G). Scale bars: 20 µm.

Article Snippet: Antibodies against the following proteins were used for immunoblotting (IB) and immunofluorescence (IF): PMCA (mouse; Novus Biologicals, Centennial, CO; cat. #NB300-578); zonula occludens (ZO1, also known as TJP1; mouse; Invitrogen; Cat #339194); Na + /K + -ATPase (mouse; Sigma-Aldrich; cat. #A-276); NCX (mouse; Thermo Fisher Scientific; cat. #MA3-926) and CSR (mouse; Cell Signaling Technology, Danvers, MA; cat, #73303; andrabbit; Thermo Fisher Scientific; cat. #PA1-934A), megalin (rabbit; Proteintech, Rosemont, IL; Cat #19700-1-AP) and NBCe1 (rabbit; Proteintech, Rosemont, IL; Cat #11885-1-AP).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Immunofluorescence, Staining, Marker, Imaging, Two Tailed Test

Journal: Applied Microbiology and Biotechnology

Article Title: Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

doi: 10.1007/s00253-023-12433-3

Figure Lengend Snippet: IFA determination of mAb 5F10 targeting NP protein in 293T cells. At 36 h post-transfection with plasmids pHW2000-QD1-NP and pHW2000, 293T cells were fixed and then cultured with primary antibody of mAb 5F10, QD1 virus-positive mouse antiserum, or the mouse (G3A1) mAb IgG1 isotype control. After incubation with the goat anti-mouse FITC-conjugated secondary antibody and then the 4′,6-diamidino-2-phenylindole (DAPI) for nucleus staining, the 293T cells were observed under fluorescence microscopy. The cells treated with the mouse antisera were used for positive control, while the cells transfected with pHW2000 empty vector or incubated with the commercial IgG1 isotype were used for negative control. Representative pictures were taken with the scale bar of 100 µm

Article Snippet: On the other hand, the sections were overnight incubated with mAb 5F10 as the primary antibody at 4 °C before incubation with the HRP-coupled goat anti-mouse secondary antibody (#CW0102, CWBIO, China) at 37 °C for 40 min. Those prepared sections were then scanned by the Pannoramic DESK slide scanner (3DHISTECH, Hungary).

Techniques: Transfection, Cell Culture, Virus, Control, Incubation, Staining, Fluorescence, Microscopy, Positive Control, Plasmid Preparation, Negative Control

Journal: Applied Microbiology and Biotechnology

Article Title: Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

doi: 10.1007/s00253-023-12433-3

Figure Lengend Snippet: Western blot analysis of the mAb 5F10 against NP protein. 293T cells transfected with empty pHW2000 vector or recombinant pHW2000-QD1-NP plasmid were lysed at 36 h post-transfection. The mAb 5F10 or IgG1 isotype and HRP-coupled goat anti-mouse IgG were served as primary and secondary antibodies, respectively. Incubation with mAb 5F10 produced a specific immunoreactive band in the lane marked with pHW2000-QD1-NP. The band was localized at about 55 kD, corresponding to the size of IAV NP protein. Treatment with an IgG1 isotype control antibody had no effect in generating positive bands. The housekeeping protein β-Actin was chosen as the internal reference

Article Snippet: On the other hand, the sections were overnight incubated with mAb 5F10 as the primary antibody at 4 °C before incubation with the HRP-coupled goat anti-mouse secondary antibody (#CW0102, CWBIO, China) at 37 °C for 40 min. Those prepared sections were then scanned by the Pannoramic DESK slide scanner (3DHISTECH, Hungary).

Techniques: Western Blot, Transfection, Plasmid Preparation, Recombinant, Incubation, Produced, Control

Journal: Applied Microbiology and Biotechnology

Article Title: Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

doi: 10.1007/s00253-023-12433-3

Figure Lengend Snippet: The broad-spectrum reactivity of mAb 5F10 against different hosts and subtypes of IAV-infected MDCK cells analyzed through IFA ( A ) and western blotting ( B ). MDCK cells were infected with avian H1~H11 IAV subtypes including H5Nx strains of multiple HA clades and NA subtypes, swine and human H1N1 and H3N2 IAV subtypes, and human type B influenza (FluB) viruses of Victoria and Yamagata lineages, respectively. Cells mock-inoculated with PBS were served as negative control. At 24 h post-infection, the infected cells were all subjected to incubation with mAb 5F10 as the primary antibody. An IgG1 isotype control antibody was simultaneously used for comparison. In the IFA analysis, typical images were taken with the scale bar of 100 µm after treatment with the FITC-conjugated goat anti-mouse IgG secondary antibody. In the western blot analysis, the HRP-conjugated goat anti-mouse IgG secondary antibody was applied to detect the immunoreactive protein bands

Article Snippet: On the other hand, the sections were overnight incubated with mAb 5F10 as the primary antibody at 4 °C before incubation with the HRP-coupled goat anti-mouse secondary antibody (#CW0102, CWBIO, China) at 37 °C for 40 min. Those prepared sections were then scanned by the Pannoramic DESK slide scanner (3DHISTECH, Hungary).

Techniques: Infection, Western Blot, Negative Control, Incubation, Control, Comparison

Journal: Applied Microbiology and Biotechnology

Article Title: Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

doi: 10.1007/s00253-023-12433-3

Figure Lengend Snippet: Conformation and conservation analysis of the epitope recognized by the anti-NP mAb 5F10. A Location of the identified epitope on NP monomer. The NP monomer structure (PDB: 7NT8) was visualized and analyzed with PyMOL software. The epitope recognized by 5F10 was labeled in red, and it locates in a protruding loop region and on the protein surface. B Sequence conservation analysis of the identified epitope. The IAV NP sequences of all sources ( n =13,166) and specifically of avian ( n =7778), swine ( n =838), and human ( n =3015) origins were retrieved from GISAID EpiFlu database with the keywords “Type”= A and “Location”= China. The dataset was collected on 7 September 2022. The variation of the epitope was analyzed with WebLogo program, and the conservation degree of each amino acid was reflected by the size of corresponding character. *Total means of the analyzed NP sequences were of all host origins including avian, swine, human, canine, and equine

Article Snippet: On the other hand, the sections were overnight incubated with mAb 5F10 as the primary antibody at 4 °C before incubation with the HRP-coupled goat anti-mouse secondary antibody (#CW0102, CWBIO, China) at 37 °C for 40 min. Those prepared sections were then scanned by the Pannoramic DESK slide scanner (3DHISTECH, Hungary).

Techniques: Software, Labeling, Sequencing

Journal: Applied Microbiology and Biotechnology

Article Title: Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus

doi: 10.1007/s00253-023-12433-3

Figure Lengend Snippet: Application of mAb 5F10. A Intracellular localization of IAV NP protein analyzed via confocal microscopy. The CEF cells were infected with a H5N6 or a H3N2 subtype IAV at 1 MOI for 12 h. B Reaction of mAb 5F10 with IAV NP protein by IP assay. MDCK cells were infected with IAV subtypes H3N2 and H5N6 at 1 MOI, and were harvested and lysed after culturing for 24 h. The lysates were then subjected to the IP assay with mAb 5F10. C Detection of IAV antigen in the lung tissue of infected mice through IHC analysis. Six-week-old female BALB/c mice were intranasally infected with a H5N6 or a H3N2 subtype IAV at 10 6.0 EID 50 , with mock-treated (with PBS) mice served as the negative control. The mice lungs were collected on day 3 post-infection for IHC staining, with mAb 5F10 serving as the primary antibody. Histopathological examination of the HE-stained sections was correspondingly performed, with the H5N6 virus induced severe lung tissue injury and atrophy of the alveoli whereas the H3N2 virus caused a relatively mild lung tissue destruction. Blue arrows indicated representative inflammatory cell infiltration, while brown signals indicated positive IHC staining. Scale bars were shown in the upper-left corner of microscopic images, with 200 µm for core images and 20 µm for zoom-in images

Article Snippet: On the other hand, the sections were overnight incubated with mAb 5F10 as the primary antibody at 4 °C before incubation with the HRP-coupled goat anti-mouse secondary antibody (#CW0102, CWBIO, China) at 37 °C for 40 min. Those prepared sections were then scanned by the Pannoramic DESK slide scanner (3DHISTECH, Hungary).

Techniques: Confocal Microscopy, Infection, Negative Control, Immunohistochemistry, Staining, Virus