Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I from Aster koraiensis inhibits SARS-CoV-2 infection. (A) The effects of various plant extracts on the entry of SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2 + and ACE2/TMPRSS2 + H1299 cells. (B, C) pSARS-CoV-2 entry and cell viability assays for the 70% EtOH extract of Aster koraiensis (B) and triterpenoid saponins isolated from A. koraiensis (C) in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data from pSARS-CoV-2 entry assay were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; **P < 0.01, ****P < 0.0001.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Isolation

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I isolated from Aster koraiensis blocks viral membrane fusion with the host cell membrane. (A) Schematic illustration of the binding of SARS-CoV-2 spike receptor binding domain (RBD) fused to GFP (RBD-GFP) to ACE2 protein overexpressed in H1299 cells. (B) Examination of the effect of AK extract and AI on the interaction between RBD-GFP and ACE2 on the surface of H1299 cells by flow cytometry after treatment with indicated concentration of AK extract and AI. The grey peaks indicate the control experiments without RBD-GFP addition. (C) ACE2 + H1299 cells were treated with 1 or 5 μM AI for 1 h and washed with fresh media (Wash) or maintained (No wash), followed by the addition of pSARS-CoV-2. The data from pSARS-CoV-2 entry assay were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (NS, not significant). (D) Schematic illustration of the SARS-CoV-2 S protein-mediated cell fusion. Addition of the cell suspension of H1299 cells stably expressing S protein and GFP (Spike-H1299) to a monolayer of ACE2/TMPRSS2 + H1299 cells with mRuby fluorescence (ACE2/TMPRSS2-H1299) leads to cell-cell fusion. (E) Still images at different time points from time-lapse imaging of S-mediated cell fusion. (F) The fusion between Spike-H1299 and ACE2/TMPRSS2 + H1299 was determined by counting the number of cells double-positive for GFP and mRuby by flow cytometry. H1299 cells expressing only GFP (no spike) were used for control experiment. All compounds were used at the concentration of 10 μM. The data were representative of three independent experiments. (G, H) Filipin cholesterol staining of ACE2 + after treatment with DMSO, AI (5 μM), and other indicated compounds (10 μM) for 1 h (G) and quantification of the intensity of membrane filipin staining using Image J software (n = 20 for each group). Error bars in the graphs indicate the SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test. ****P < 0.0001; NS not significant (H).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Isolation, Binding Assay, Flow Cytometry, Concentration Assay, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Imaging, Staining, Software

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I prevents SARS-CoV-2 S-induced syncytia formation. (A) Schematic illustration of Split-GFP assay. Ectopic expression of SARS-CoV-2 spike protein into cultures of the mixture of ACE2/TMPRSS2 + H1299 cells expressing GFP1-10 or GFP11 leads to cell-cell fusion, generating GFP fluorescence. (B) Images of the S-mediated cell-to-cell fusion using Split-GFP. GFP and blue fluorescence indicate the cell-to-cell fusion and DAPI-stained nuclei, respectively. The nuclei are automatically pseudocolored in white and overlapped with GFP fluorescence by CellReporterXpress software. (C) Examination of effect of AI on the S-mediated cell-cell fusion using Split-GFP assay. Representative images of GFP-positive cell-to-cell fusion. The GFP signal and DAPI nuclei staining are automatically pseudocolored in green and red by CellReporterXpress software. (D) Quantitative evaluation for cell-cell fusion using Split-GFP assay. Images of GFP and nuclei stained with DAPI were obtained in five random fields per well. The percentage of fusion cells were calculated by dividing number of nuclei in GFP-positive cells by total number of nuclei. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; ****P < 0.0001; NS not significant. (E) Protein lysates from co-culture experiments were assessed by Western blot. GAPDH was used as a loading control.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Split GFP Assay, Expressing, Fluorescence, Staining, Software, Co-Culture Assay, Western Blot

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I equally inhibits the infection of SARS-CoV-2 WT and D614G mutant. (A) pSARS-CoV-2 entry assay in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (****P < 0.0001). (B) The effects of AI on the infection of WT and D614G mutant of pSARS-CoV-2 in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Mutagenesis, Two Tailed Test

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I inhibits the infection of SARS-CoV-2 and its variants with a similar efficiency. (A, C) Dose-response curves for AI (A) and chloroquine (C) against ancestral SARS-CoV-2 (A, black circle) and SARS-CoV-2 variants Alpha (B.1.1.7, red square), Beta (B.1.351, orange triangle), Delta (B.1.617.2, pink inverted triangle), and Omicron (B.1.1.529, green diamond) in Vero cells. The blue diamond represents cell viability. The mean ± SEM was calculated from duplicate experiments. (B, D) Confocal images of SARS-CoV-2 N protein (green) and cell nuclei (de Vries et al.) at concentrations near the IC 50 of AI (B) and chloroquine (D) in Vero cells. (E) Dose-response curve for AI against replicable SARS-CoV-2 recombinant viruses expressing Nanoluciferase (Nluc) into A549 cells that overexpress ACE2 and TMPRSS2. The blue diamond represents cell viability.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Recombinant, Expressing

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Knockdown of calcineurin expression in T cells recapitulates the effects of the drugs. (a,b) Top, representative immunoblot analysis for the expression of calcineurin in lysates from Jurkat T cells (a) or human CD4+ T cells (b) that were untransfected or electroporated with a control (Ctrl), calcineurin A α-specific, or calcineurin A β-specific siRNA either individually or in combination 72 h after transfection. Bottom, quantification of calcineurin expression relative to ZAP70 levels. In b, the lanes were rearranged for clarity. (c,d) Representative western blot analysis of lysates from siRNA-treated Jurkat cells (c) and human CD4+ T cells (d) after stimulation with OKT3 and anti-mouse-IgG for the indicated times. Stripped membranes were reblotted to ascertain total amounts of the indicated cellular proteins. Data are representative of three independent experiments. (e) Immunoblot analysis of Jurkat cells expressing endogenous or endogenous plus codon-optimized calcineurin A (calcineurin Aopt) after activation with OKT3 for 5 min. The data are representative of three (c,d) or two (e) independent experiments with similar results.

Article Snippet: A plasmid expressing codon-optimized calcineurin A (SFG.wtCNa_opt.IRES.eGFP), in which each of the three siRNA-targeted sequences containing multiple codons were altered from that of the wild-type sequence (while maintaining the same amino acid sequence), was obtained from Addgene (#22489). pSX SRα, encoding human ZAP70 cDNA has been described 41 .

Techniques: Expressing, Western Blot, Transfection, Activation Assay

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Cytosolic calcineurin is recruited to TCR microclusters. (a,b) Immunostained images of Jurkat cells stably expressing YFP-tagged ZAP70 that were dropped onto coverslips coated with anti-CD3 (stimulatory; top) or anti-CD45 (nonstimulatory; bottom) for 2.5 min (a) or 10 min (b). ZAP–YFP is shown in green, anti-calcineurin-A is shown in red, and phosphotyrosine is shown in blue. The experiment was performed 3 times, and in each case a representative single confocal slice is shown. Scale bars, 5 μm.

Article Snippet: A plasmid expressing codon-optimized calcineurin A (SFG.wtCNa_opt.IRES.eGFP), in which each of the three siRNA-targeted sequences containing multiple codons were altered from that of the wild-type sequence (while maintaining the same amino acid sequence), was obtained from Addgene (#22489). pSX SRα, encoding human ZAP70 cDNA has been described 41 .

Techniques: Stable Transfection, Expressing

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Calcineurin-containing microclusters also contain phosphorylated TCR. Immunostaining of (a,b) Immunostained images of Jurkat cells stably expressing YFP-tagged ZAP70 that were dropped onto anti-CD3-coated (stimulatory) or anti-CD45-coated (nonstimulatory) coverslips for 2.5 min (a) or 10 min (b). ZAP–YFP is shown in green, anti-calcineurin-A is shown in red, and phospho-TCR-ζ is shown in blue. The experiment was performed 3 times, and in each case a representative single confocal slice is shown. Scale bars, 5 μm.

Article Snippet: A plasmid expressing codon-optimized calcineurin A (SFG.wtCNa_opt.IRES.eGFP), in which each of the three siRNA-targeted sequences containing multiple codons were altered from that of the wild-type sequence (while maintaining the same amino acid sequence), was obtained from Addgene (#22489). pSX SRα, encoding human ZAP70 cDNA has been described 41 .

Techniques: Immunostaining, Stable Transfection, Expressing

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Association of calcineurin with signaling components requires LCK activity and intact ZAP70. (a,b) Immunoblot analysis for co-immunoprecipiation of calcineurin after immunoprecipitation of TCR-ζ from activated Jurkat cells (a) and human CD4+ T cells (b) that were treated as indicated (with or without FK506 or CsA) and stimulated with anti-CD3 for the indicated times. (c) Immunoblot analysis of TCR-ζ immunoprecipitates from Jurkat cells that were incubated with or without the Src kinase family inhibitor PP1 and activated for different amounts of times. Whole-cell lysates (WCL) from these cells were subjected to immunoblot analysis with a phospho-LCKY394-specific antibody to confirm inhibition of kinase activity. (d,e) Immunoblot analysis of anti-calcineurin immunoprecipitates from Jurkat cells (d) and human CD4+ T cells (e) that were activated for the indicated times. Stripped membranes were reprobed with anti-calcineurin to ensure equal amounts of loading of the precipitated complex. (f,g) Immunoblotting of anti-TCR-ζ immunoprecipitates from Jurkat cells and ZAP70-deficient P116 cells (f) or from Jurkat cells that were or were not treated with the ZAP70 kinase inhibitor piceatannol (g) following activation for different amounts of times. Whole-cell lysates were subjected to immunoblot analysis with anti-phospho-LATY171 to confirm inhibition of ZAP70 activity. (h,i) Immunoblot analysis of anti-calcineurin-A (or an isotype control) (h) or anti-TCR-ζ (i) immunoprecipitates from Jurkat cells that were unactivated or activated for the indicated amounts of times. The data in a–i are representative of three independent experiments with similar results.

Article Snippet: A plasmid expressing codon-optimized calcineurin A (SFG.wtCNa_opt.IRES.eGFP), in which each of the three siRNA-targeted sequences containing multiple codons were altered from that of the wild-type sequence (while maintaining the same amino acid sequence), was obtained from Addgene (#22489). pSX SRα, encoding human ZAP70 cDNA has been described 41 .

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Incubation, Inhibition, Activation Assay

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: phospho-LCKS59 is a substrate of calcineurin in vivo and in vitro. (a,b) Immunoblot analysis of Jurkat cells (a) and primary human CD4+ T cells (b) that were treated with either medium or the indicated compounds and stimulated with anti-CD3 for the indicated amounts of time. (c) Immunoblot analysis of anti-TCR-ζ immunoprecipitates from Jurkat cells after activation for the indicated amounts of time. (d,e) Immunoblot analysis of proteins from Jurkat cells (d) and human CD4+ T cells (e) that were transfected with a control siRNA or with PPP3CA-specific and PPP3CB-specific siRNAs and that were activated with anti-CD3 for the indicated amounts of time. (f) Immunoblot analysis of TCR-ζ immune-complexes from Jurkat cells that were activated in the absence or presence of FK506 for the indicated times and subjected to an in vitro phosphatase assay using recombinant human calcineurin (rhCN). The data in a–f are representative of three independent experiments.

Article Snippet: A plasmid expressing codon-optimized calcineurin A (SFG.wtCNa_opt.IRES.eGFP), in which each of the three siRNA-targeted sequences containing multiple codons were altered from that of the wild-type sequence (while maintaining the same amino acid sequence), was obtained from Addgene (#22489). pSX SRα, encoding human ZAP70 cDNA has been described 41 .

Techniques: In Vivo, In Vitro, Western Blot, Activation Assay, Transfection, Phosphatase Assay, Recombinant