Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Plasmid Preparation, Binding Assay, Flow Cytometry, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay

Journal: Cancer biology & therapy

Article Title: Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice.

doi: 10.4161/cbt.7.3.5425

Figure Lengend Snippet: Figure 2. Characterization of anti-4-1BB scFv expression. (A) RT-PCR analy- sis of anti-4-1BB scFv mRNA expression. Cultured cells were infected with Ad.CMV.AP or Ad.4-1BB scFv at a MOI of 10 for 24 h. The transcripts of anti- 4-1BB scFv were semi-quantified by RT-PCR with β-actin as an internal control. (B) Flow cytometric analysis of anti-4-1BB scFv cell surface expression. Hepa 1-6 cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 10 for 24 h and stained with FITC-conjugated anti-human IgG F(c) antibody that recognized the immunoglobulin Fc fragments expressed on Ad.4-1BB scFv infected Hepa1-6 cells for flow cytometry analysis. (C) Visualizing anti-4-1BB scFv expression on the surface of Hepa1-6 cells by direct immunofluorescence staining. Cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 30 for 24 h, fixed, incubated with FITC-conjugated anti-human IgG F(c) anti- body, and counterstained with Hoechst 33342. The same optical field was photographed under fluorescence light and normal light. The blue fluores- cence corresponded to cell nuclei, and the green fluorescence corresponded to anti-4-1BB scFv-hIgG Fc. Original magnification, x630.

Article Snippet: After 24 h of infection, cells were harvested, washed twice with ice-cold PBS and incubated in a 1:250 dilution of a fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG F(c) (Rockland Immunochemicals Inc., PA, USA) on ice for 1 h, which recognized the immunoglobulin Fc fragments expressed on virus infected Hepa1-6 cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Infection, Control, Staining, Flow Cytometry, Immunofluorescence, Incubation, Fluorescence