ace2 spike binding inhibition activity  (BMG Labtech)

Journal: bioRxiv

Article Title: Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation In Airway Epithelial Cells

doi: 10.1101/2022.03.18.484956

Figure Lengend Snippet: (A) Attached SARS-CoV-2 virions on the cell surface were detected by RT-qPCR. Calu-3 cells were pretreated with Gal-9 for one hour, then cells were incubated with SARS-CoV-2 (MOI=0.01) in solutions with or without Gal-9 at the indicated concentrations at 4°C for two hours. Cells were washed three times with PBS and harvested for RNA isolation and RT-qPCR measurement of SARS-CoV-2 N gene expression. (B) Relative infectivity of SARS-2-S pseudotyped virus and VSV-G pseudotyped virus in Calu-3 treated with Gal-9 at the indicated concentrations. Calu-3 cells were exposed to Gal-9 for six hours and then infected with SARS-2-S pseudotyped virus or VSV-G pseudotyped virus in solutions containing Gal-9 at the indicated concentrations. Pseudotyped viral entry was analyzed by luciferase activity 24 hpi. Positive serum predetermined to possess anti-SARS-CoV-2 neutralizing activity was used as a negative control. Luciferase signals obtained in the absence of Gal-9 were used for normalization. (C) Relative infectivity of SARS-2-S pseudotyped virus and VSV-G pseudotyped virus in Calu-3 cells treated with anti-ACE2 Ab at the indicated concentrations. Calu-3 cells pre-incubated with anti-ACE2 Ab (R&D Systems, AF933) at the indicated concentrations, or control antibody (anti-goat IgG (R&D Systems, AB-108-C), 50 μg/ml) were co-administered with SARS-2-S pseudotyped and VSV-G pseudotyped virus. At 24 hpi, pseudotyped viral entry was analyzed by luciferase activity. Luciferase signals obtained using the control antibody (anti-goat IgG, 50 μg/ml) were used for normalization. (D) The effect of anti-ACE2 Ab on Gal-9-enhanced cell entry of SARS-2-S was evaluated by measuring luciferase activity. Calu-3 cells were pretreated with Gal-9 for six hours and then pre-incubated with anti-ACE2 Ab (25 μg/ml) or control antibody for one hour, and cells were inoculated with SARS-2-S pseudotyped or VSV-G pseudotyped virus in a solution containing Gal-9 at the indicated concentrations. At 24 hpi, pseudotyped viral entry was analyzed by luciferase activity. Luciferase signals obtained in the absence of both Gal-9 and anti-ACE2 Ab were used for normalization. Data are representative of the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by t test. p>0.05 [ns], p≤0.05 [*], p≤ 0.01 [**], p≤0.001 [***], p≤0.0001 [****].

Article Snippet: Assessment of Gal-9 effects on the binding of the SARS-CoV-2 spike to human ACE2 was performed using a commercially available spike-ACE2 binding assay kit (CoviDrop SARS-CoV-2 Spike-ACE2 Binding Activity/Inhibition Assay Kit, EPIGENTEK, D-1005-48) following the protocol provided by the manufacturer.

Techniques: Quantitative RT-PCR, Incubation, Isolation, Expressing, Infection, Luciferase, Activity Assay, Negative Control

Journal: bioRxiv

Article Title: Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation In Airway Epithelial Cells

doi: 10.1101/2022.03.18.484956

Figure Lengend Snippet: (A) Representative flow cytometry plot describing the protein levels of ACE2 and TMPRSS2 on the surface of Calu-3 cells treated with Gal-9. Calu-3 cells were treated with Gal-9 at the indicated concentrations for 24 h. Cells were then washed and detached before antibody staining for flow cytometry. (B) Percentages of cells expressing ACE2 or TMPRSS2 at the cell surface, measured using flow cytometry. Data represent the results of three independent experiments (mean ± SEM). (C) The dose-response of SARS-CoV-2 spike-ACE2 binding activity measured by reading the absorbance at the wavelength of 450 nm. Data represent the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by t test. p>0.05 [ns], p ≤ 0.05 [*], p ≤ 0.01 [**], p ≤ 0.001 [***], p ≤ 0.0001 [****].

Article Snippet: Assessment of Gal-9 effects on the binding of the SARS-CoV-2 spike to human ACE2 was performed using a commercially available spike-ACE2 binding assay kit (CoviDrop SARS-CoV-2 Spike-ACE2 Binding Activity/Inhibition Assay Kit, EPIGENTEK, D-1005-48) following the protocol provided by the manufacturer.

Techniques: Flow Cytometry, Staining, Expressing, Binding Assay, Activity Assay