Journal: Pharmaceuticals

Article Title: In Vitro Effect of Taraxacum officinale Leaf Aqueous Extract on the Interaction between ACE2 Cell Surface Receptor and SARS-CoV-2 Spike Protein D614 and Four Mutants

doi: 10.3390/ph14101055

Figure Lengend Snippet: Binding inhibition of S1 spike protein to human HEK293-hACE2 cells by extract pre-incubation. Cells were pre-incubated for the indicated times with 10 mg/mL T. officinale (TO), its HMW fraction, equal to 10 mg/mL extract (HMW), and 10 mg/mL C. intybus (CI) or solvent control (a.d.) and subsequently treated with His-tagged S1 spike protein for 1 h without a washing step in between at 4 °C. Binding inhibition was assessed using flow cytometry. N = 3, bars are means + SD. Upper left: cytogram of gated HEK-hACE2 cells. Middle: overlay of representative fluorescence intensity histograms for ACE2 surface expression. Upper right: overlay of representative fluorescence intensity histograms for spike-binding inhibition by the extracts or a.d.; positive control: 20 µg/mL soluble hACE2. Cells were stained with anti-His-tag Alexa Fluor 647 conjugated monoclonal antibody; ** p < 0.01. Significance of difference was calculated relative to the solvent control by one-way ANOVA.

Article Snippet: Cells were then washed one time with PBS buffer containing 1% FBS at 500g, 5 min before staining with His-tag A647 mAb (Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) for 30 min at RT.

Techniques: Binding Assay, Inhibition, Incubation, Solvent, Flow Cytometry, Fluorescence, Expressing, Positive Control, Staining

Journal: Pharmaceuticals

Article Title: In Vitro Effect of Taraxacum officinale Leaf Aqueous Extract on the Interaction between ACE2 Cell Surface Receptor and SARS-CoV-2 Spike Protein D614 and Four Mutants

doi: 10.3390/ph14101055

Figure Lengend Snippet: Binding inhibition of spike D614, and its mutants D614G, N501Y or mix (N501Y, K417N and E484K) to human HEK293-hACE2 and A549-hACE2-TMPRSS2 cells by extract pre- or post-incubation. Overlay of fluorescence intensity histogram for ( A ) unstained HEK cells, staining control (anti-His-tag Alexa Fluor 647), and cells incubated with His-tag-labelled spike D614, D614G or N501Y for 1 h at 4 °C. ( B , C ) cells pre-incubated with solvent control (a.d.), 10 mg/mL T. officinale (TO) or 10 mg/mL C. intybus (CI) for 30–60 s, and then treated with His-tag-labelled S1 spike D614, D614G or N501Y protein for 1 h without a washing step in between at 4 °C. ( D – G ) Effect of extract incubation on HEK or A549 cells either before or after incubation with His-tag-labelled spike D614, D614G, N501Y or mix (N501Y, K417N and E484K) protein at 37 °C. ( H ) Plant extracts were incubated in saliva from four human donors for 0.5 h at 37 °C. Afterwards, cells were pre-treated with 5 mg/mL extracts for 60 s at 37 °C before incubation with His-tag-labelled spike D614 protein for 0.5 h at 37 °C. Spike-binding inhibition to human cells was assessed using flow cytometric analysis of cells stained with anti-His-tag Alexa Fluor 647 conjugated monoclonal antibody. Bars are means +SD; * p < 0.05, ** p < 0.01. Significance of difference was calculated relative to the respective solvent control by one-way ANOVA.

Article Snippet: Cells were then washed one time with PBS buffer containing 1% FBS at 500g, 5 min before staining with His-tag A647 mAb (Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) for 30 min at RT.

Techniques: Binding Assay, Inhibition, Incubation, Fluorescence, Staining, Solvent