Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Upregulation of the UII system in the kidneys of neonatal pigs following IR injury. Quantitative RT-PCR analysis showing relative mRNA expression levels of (A) furin, (B) UII, (C) URP, and (D) UT in whole kidney tissue, and (E) UT in intrarenal arteries from sham and IR piglets ( n = 4 per group). (F–G) Representative Western blot and densitometric analysis of UT protein expression in intrarenal arteries ( n = 4 per group). (H–I) Western blot and quantification of furin protein expression in kidney tissues ( n = 4 per group). (J–K) representative immunohistochemistry (IHC) images and quantification of UII immunostaining intensity in renal tubules from sham and IR piglets (4 kidney sections per group). * p < 0.05, unpaired t -test.

Article Snippet: For Western blot analysis, the primary antibodies included a rabbit polyclonal anti-furin antibody (18413-1-AP, Proteintech, Rosemont, IL, USA) at a 1:500 dilution and a rabbit polyclonal anti-GPR14 (UT receptor) antibody (TA358466, OriGene Technologies, Rockville, MD, USA) at a 1:100 dilution.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Immunostaining

Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Furin inhibition reduces cIR-driven UII production, and UT and furin blockade attenuate cIR-induced cytotoxicity in primary neonatal pig proximal tubule epithelial cells. (A) Representative fluorescence microscopy images showing furin and UII immunostaining and their colocalization in primary neonatal pig proximal tubule epithelial cells (PTECs; 3 biological replicates). (B) Quantification of intracellular furin levels in control (ctrl) and chemical ischemia-reperfusion (cIR)–treated cells ( n = 4). (C) Secreted UII levels in cIR-treated cells in the absence or presence of the furin inhibitor SSM3 trifluoroacetate (SSM3; n = 4). (D) cIR-induced cytotoxicity was significantly reduced by pharmacological inhibition of furin (SSM3) and by UT inhibition with urantide (URTD). * p < 0.05 ctrl vs. cIR; p < 0.05 cIR vs. SSM3 + cIR; $ p < 0.05 cIR vs. both SSM3 + cIR and URTD + cIR. Unpaired t -test (B); One-way ANOVA and Holm-Šídák’s multiple comparisons tests (C–D); n = 4, each. Scale bar = 50 µm.

Article Snippet: For Western blot analysis, the primary antibodies included a rabbit polyclonal anti-furin antibody (18413-1-AP, Proteintech, Rosemont, IL, USA) at a 1:500 dilution and a rabbit polyclonal anti-GPR14 (UT receptor) antibody (TA358466, OriGene Technologies, Rockville, MD, USA) at a 1:100 dilution.

Techniques: Inhibition, Fluorescence, Microscopy, Immunostaining, Control

Journal: JCI Insight

Article Title: C-terminal FGF-23 production coupling with aldosterone via FAM20C and predicting cardiovascular events in primary aldosteronism

doi: 10.1172/jci.insight.166461

Figure Lengend Snippet: ( A ) The dose-dependent effect of aldosterone on the upregulation of cFGF-23, n = 4/group. ( B ) The upregulation of cFGF-23 after aldosterone treatment remains unaffected by the furin inhibitor, n = 3/group. ( C ) Silencing Fam20C (shRNA) effectively restored the upregulated cleavage of cFGF-23, n = 3/group. Statistical analysis was conducted using 1-way ANOVA with Bonferroni’s post hoc correction for multiple comparisons (* P < 0.05, vs. control group). The graph data are presented as mean ± SEM.

Article Snippet: For Western blot analysis, polyclonal anti-furin antibody (sc-20801, obtained from Santa Cruz Biotechnology); mouse monoclonal antibodies targeting iFGF-23 and cFGF-23 (MAB26291, acquired from R&D Systems, Bio-Techne), with the distinction between the two made based on their molecular sizes on a gel; rabbit polyclonal FAM20C antibody (25395-1-AP, sourced from Proteintech); and rabbit polyclonal anti-GAPDH antibody (MAB374, obtained from MilliporeSigma) were utilized.

Techniques: shRNA, Control

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: List of the oligonucleotides used for the host gene and cytokine expression profiling.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The host Furin as a potential novel target for the bovine miRNA16a. ( A ) In silico prediction of miRNA16a targeting the 3’ UTR of the host cell Furin. The context + + score percentile represents the binding energy of the miRNA with the target gene. ( B ) Multiple sequence alignment shows that the miRNA16a/Furin binding region (indicated in the red color font boxes) is conserved among eight species, including humans, mice, and bovines. ( C ) The MDBK cells were transfected with either miR-Scr or miRNA16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h, the samples were subjected to qRT‒PCR. ( D ) Western blot analysis of host Furin protein expression in the MDBK cells in the miR-Scr- and miRNA16a-transfected groups. ( E ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( F ) The BEC cells were transfected with scrambled and miRNA16a, followed by BCoV enteric and respiratory isolate infection. After 72 h, samples were collected for the qRT-PCR analysis to assess the mRNA expression level of the host Furin in both the scrambled- and miRNA16a-transfected groups. ( G ) Western blot analysis of host Furin protein expression in the scrambled- and miRNA16aa-transfected groups of BEC cells. ( H ) Western blot band density of Furin protein normalized to that of β-actin in BECs. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: In Silico, Binding Assay, Sequencing, Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Validation of the host cell furin as a functional target for the host cell miRNA16a. ( A ) The MDBK cells were transfected independently with either miR-Scr or Antagomir-16a, followed by BCoV/Ent or BCoV/Resp infection. After 72 h of transfection, the collected samples were subjected to qRT-PCR to evaluate the host cell furin mRNA expression levels. ( B ) Western blot analysis of host Furin protein and β-actin expression in the MDBK cells in the miR-Scr- and Antagomir-16a-transfected groups. ( C ) Western blot band density of Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The results of the dual-luciferase reporter assay. HEK cells were transfected with different combinations of the Furin 3’UTR (WT) or mutant (Mut) plasmid or vector only (pmir-GLo) with miRNA16a, Antagomir-16a, and miR-Scr, as indicated. The dual luciferase assay was conducted, and the relative luciferase activity was calculated using red firefly luciferase signals as a normalization control for the green Renilla luciferase signals. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Biomarker Discovery, Functional Assay, Transfection, Infection, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Mutagenesis, Plasmid Preparation, Activity Assay, Control, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The impacts of silencing the host Furin and BCoV-S glycoproteins by specifically designed siRNA molecules on the BCoV replication. ( A ) Schematic representation of the siRNA transfection BCoV infection experiments: the transfection process of the scrambled-siRNA, siRNA-Furin, and the siRNA-BCoV-S glycoproteins into the MDBK the BEC cells was illustrated, followed by infection with independent BCoV/Ent or BCoV/Resp isolates infection in these cells for 72 h and subsequent sample collection for further analysis. ( B ) The MDBK cells were transfected with 50 ng or 100 ng of the siRNA-Furin, followed by western blot analysis of Furin protein expression levels. ( C ) Western blot band density of the Furin protein normalized to that of β-actin in the MDBK cells. ( D ) The MDBK cells were transfected with 50 ng or 100 ng of siRNA BCoV/-S, followed by western blot analysis of BCoV/S protein expression. ( E ) Results of the western blot band density of the BCoV/S glycoprotein normalized to that of β-actin in the MDBK cells. ( F ) The qRT-PCR results show the Furin mRNA expression levels in the scrambled siRNA- and the siRNA-Furin-transfected MDBK cells ( G ) and BECs. ( H ) The qRT-PCR results analysis shows the genome copy numbers of the BCoV in the cases of the scrambled-siRNA and the siRNA-Furin-transfected MDBK cells ( I ) and BEC cells. ( J ) The qRT-PCR analysis results show the genome copy numbers of the BCoV in the case of the scrambled-siRNA and siRNA-BCoV/Spike-transfected MDBK cells ( K ) and the BEC cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Silencing of the host cell Furin protein and the BCoV-S glycoprotein negatively impacted BCoV infectivity in bovine cells. ( A ) The viral infectivity of the BCoV/Ent or BCoV/Resp was markedly inhibited in cells transfected with siRNA-BCoV/S or (B) the siRNA-Furin, as measured by the viral plaque assay. The plaque-forming units (PFUs) were used to determine the viral titer in each virus-infected group. ( C ) The MDBK cells were transfected with scrambled siRNA (Scr-siRNA), siRNA-Furin, or siRNA BCoV/-Spike, followed by infection with either the BCoV/Ent or the BCoV/Resp. Cells were lysed after 72 hpi, and western blot analysis was performed to observe the protein expression of the BCoV-S, the BCoV-N, and the host cell Furin proteins expression. ( D ) Western blot band density of the Furin protein, ( E ) BCoV spike protein, and ( F ) BCoV-N protein was normalized to that of β-actin in the MDBK cells. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Infection, Transfection, Viral Plaque Assay, Virus, Western Blot, Expressing, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Silencing of the host cell Furin and BCoV-S glycoproteins impacted the expression levels of some other coronavirus replication-related proteins (ACE2, NRP1, and TMPRSS2) which further impacted BCoV/Ent/BCoV/Resp replication. ( A ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA BCoV/Spike glycoprotein, followed by the infection with either BCoV/Ent or BCoV/Resp. Cell lysates were collected after 72 hpi and subjected to Western blot analysis to assess the protein expression levels of the bovine (ACE2, NRP1, TMPRSS2, and bovine β-actin) proteins. ( B ) Results of the qRT-PCR analysis of bovine TMPRSS2 mRNA expression levels in the case of the siRNA-Scrambled-, the siRNA-Furin-, or the siRNA-BCoV-S transfected MDBK cells. ( C ) Results of the analysis of the western blot band densities of the TMPRSS2 protein expression normalized to that of bovine β-actin protein. ( D ) Results of the qRT-PCR analysis of bovine ACE2 mRNA expression levels. ( E ) Results of the western blot band densities of the ACE2 protein normalized to that of β-actin. ( F ) Results of the qRT-PCR analysis of bovine NRP1 mRNA expression. ( G ) Results of the western blot band densities of the NRP1 protein expression levels normalized to that of β-actin. The data of all the experiments represent at least three biological repeats. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Expressing, Transfection, Control, Infection, Western Blot, Quantitative RT-PCR, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: Activation of the host cell cytokine-like Strome upon miRNA16a overexpression during BCoV infection in bovine cells. The MDBK cells were transfected with either Scr-miRNA or miRNA16a, followed by infection with BCoV/Ent or BCoV/Resp. The results. Results of the qRT-PCR analysis to quantify the mRNA expression of ( A ) IFN-α; ( B ) IFN-β; ( C ) IFN-γ; ( D ) IL-6; ( E ) and IL-10. ( F ) The MDBK cells were transfected with Scr-siRNA control, siRNA-Furin, or siRNA-BCoV-Spike, followed by BCoV/Ent or BCoV/Resp infection. The qRT-PCR analysis of the mRNA expression levels of IFN-α, ( G ) IFN-β, ( H ) IFN-γ, ( I ) IL-6, ( J ) and IL-10. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Activation Assay, Over Expression, Infection, Transfection, Quantitative RT-PCR, Expressing, Control, Comparison

Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The proposed model of the mechanism of the dual actions of the host cell miRNA16a in restricting BCoV replication through targeting the BCoV-S glycoprotein and the host cell Furin and enhancing cytokine expression. ( A ) Schematic representation of the BCoV-Spike protein, highlighting the S1 and S2 subunits and Furin cleavage site (S1/S2). The miRNA16a targets two regions within the S1 subunit, indicated in red (starting positions 24,762 and 24,928). ( B ) A schematic diagram of the bovine Furin shows the miRNA16a target sites in the 3’UTR of the bovine host cell furin; red fonts indicate the starting nucleotides of the seed region of miRNA16a (starting position 793). ( C ) Illustration of the bovine cells transfected with miRNA scrambled (miRNA-Scr) followed by BCoV infection. The BCoV viral genome is released into the cytoplasm of infected cells, after which viral replication and the production of nested sets of sg mRNAs are initiated. BCoV infection markedly inhibited the expression of some host cytokines (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10) but enhanced BCoV production. ( D ) A schematic illustration of the bovine cells transfected with miRNA16a followed by infection with BCoV. The miRNA16a targets host Furin at the cell surface, reducing the BCoV replication by abolishing the BCoV/S glycoprotein cleavage. The overexpression of miRNA16a also showed marked inhibition of the BCoV-S glycoprotein expression levels, ultimately reducing the production of other viral proteins, particularly BCoV-N. The miRNA16a, which targets BCoV-S and the host Furin, substantially inhibited the production of new BCoV progeny. Moreover, this targeting markedly increased the expression of some host cell cytokines, especially the (IFN-α, IFN-β, IFN-γ, IL-6, and IL-10), which led to further inhibition of BCoV progeny release.

Article Snippet: Furin rabbit anti-bovine polyclonal (Cat. No. ARP45328_P050), ACE2 rabbit anti-bovine polyclonal (Cat. No. ARP53751_P050), TMPRSS2 rabbit anti-bovine polyclonal (Cat. No. ARP46628_P050), and anti-NRP1 rabbit anti-bovine polyclonal (Cat. No. ARP59101_P050) were purchased from Aviva Systems Biology.

Techniques: Expressing, Transfection, Infection, Over Expression, Inhibition

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) Immunoblots showing shedded S1 subunits and human IgG heavy chain (IgG Hc), full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of antibody-treated HEK293T cells expressing WT full-length spike for 16 hours. CB6, FD20, REGN10933 and REGN10987 were diluted in PBS and used at 12.5 nM, blots are representative of at least three independent experiments; ( B ) Immunoblots showing proteinase K-resistant S2’ cleavage product, obtained from cell lysates described in (A) and were then treated in the absence or presence of 10 μg/mL proteinase K for 30 min at 37°C, blots are representative of two individual repeats; ( C ) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells. CB6 doses used were 0.1, 0.5, 2.5 and 12.5 nM respectively, human IgG isotype was used at 12.5 nM for the negative control. Data and blots are representative of four individual repeats; ( D ) Immunoblots showing shedded S1 subunits, full-length spike and S1 collected from supernatant and cell lysate fractions of lentivirus-transduced A549 cells expressing WT full-length spike, stimulated without or with 12.5 nM CB6 antibody. Blots are representative of three individual repeats; ( E ) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells expressing Beta spike VOCs carrying N417 or K417 revert mutation, blots and data are representative of four individual repeats; ( F ) Schematics of the R685A (furin-cleavage site deficient) spike mutant, and representative fluorescent images captured at 594 nm and 405 nm from HEK293T cells expressing WT and R685A spike mutant, stimulated without or with 12.5 nM CB6; scale bars are representative of 50 μm, images were representative of two individual repeats; ( G ) Luciferase activity (RLU) measured from antibody-induced cell-cell fusion assay, where co-cultured HEK293T cells expressing WT or R685A spike mutant were stimulated without or with 12.5 nM CB6 for 16 hours (Top). Supernatants and cell lysates were used for immunoblots of shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ (Bottom). Blots are representative of three independent experiments.

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Western Blot, Expressing, Luciferase, Activity Assay, Cell-Cell Fusion Assay, Negative Control, Mutagenesis, Cell Culture

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) Immunoblots of shedded S1 subunits, IgG Hc collected from supernatants; or full-length spike, S1, S2 and cleaved S2’ and IgG Hc collected from HEK293T cell lysates expressing WT or R685A spike mutant, stimulated without or with 12.5 nM CB6 antibody for 16 hours, in the absence or presence of 50 nM Bafilomycin A1 (BafA1). Blots are representative of three individual experiments; ( B ) Representative confocal images of 12.5 nM CB6 antibody-stimulated HEK293T cells expressing WT or R685A spike mutant for 16 hours. Anti-Early endosomes antigen 1 (EEA1) and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively, co-localizations are indicated with white arrows and R values derived from Pearson’s coefficients summarized, scale bars are representative of 10 μm. Images are representative of four individual experiments; ( C ) Luciferase activity (RLU) measured from 12.5 nM CB6-stimulated siControl or siFURIN donor HEK293T cells co-expressing Cre and WT spike, mixed with siControl or siFURIN acceptor Stop-Luc- expressing cells for 16 hours (top); and immunoblots showing shedded S1 subunits, hIgG Hc and full-length spike, S1, S2 and S2’ collected from co-cultured cell supernatants and lysates (bottom). Data shown are representative of four independent repeats; ( D ) Luciferase activity (RLU) measured from 12.5 nM CB6-stimulated over-expressing pcDNA4 (empty vector) or FURIN donor HEK293T cells co-expressing Cre and WT spike, mixed with over-expressing pcDNA4 or FURIN acceptor Stop-Luc -expressing cells for 16 hours (top); and immunoblots showing shedded S1 subunits, hIgG Hc and full-length spike, S1, S2 and S2’ collected from co-cultured cell supernatants and lysates (bottom). Data shown are representative of four independent repeats; ( E ) Luciferase activity (RLU) measured from HEK293T cells co-expressing WT spike and Cre, mixed with Stop-Luc -expressing cells, in the absence or presence of 25 μM dec-RVKR-cmk (RVKR) for 16 hours (top); immunoblots showing shedded S1 subunits, full-length S, S1, S2 and cleaved S2’ collected from supernatants and lysates (bottom). Data shown are representative of five independent repeats, and the blot is representative of three repeats; ( F ) Representative confocal images of 12.5 nM CB6 antibody-stimulated HEK293T cells expressing WT spike, treated with DMSO or 25 μM RVKR for 16 hours. Anti-Early endosomes antigen 1 (EEA1) and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively, scale bars are representative of 10 μm, co-localizations are indicated with white arrows and R values derived from Pearson’s coefficients were summarized. Data are displayed as individual points with mean ± standard error of the mean (SEM). P value was obtained by one-way ANOVA with Sidak’s post hoc test and is indicated on the figure.

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Western Blot, Expressing, Mutagenesis, Staining, Derivative Assay, Luciferase, Activity Assay, Cell Culture, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) When SARS-CoV-2 spike is cleaved at the S1/S2 cleavage site and expressed on the infected cell membrane, binding of Class I antibodies (Abs) onto the receptor binding motif (RBM) trigger the rapid shedding of S1 subunit at the cell surface. This event allows the functional activation of the S2’ cleavage site by membrane bound proteases, such as TMPRSS2. Exposure of fusion peptide at the plasma membrane triggers receptor-independent cell-cell fusion among adjacent cells. This process drives the functional activation of spike-expressed on the cell membrane and promote cell-cell transmission of the SARS-CoV-2 virus; ( B ) However, when spike S1/S2 site is not cleaved by an endogenously expressed protease, for instance by host cell furin or TMPRSS2, binding of Class I Abs on the RBM is unable to trigger S1 shedding from the spike-expressing cells. Instead, binding of Class I Ab leads to the internalization of spike trimers, where S2’ cleavage and exposure of fusion peptide could occur inside endolysosomes. As a result, cell-cell transmission of the SARS-CoV-2 virus could be efficiently prevented. Figure was illustrated using images created with BioRender.com .

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Infection, Membrane, Binding Assay, Functional Assay, Activation Assay, Clinical Proteomics, Transmission Assay, Virus, Expressing