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apc conjugated antibody against integrin β6  (R&D Systems)


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    R&D Systems apc conjugated antibody against integrin β6
    a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human <t>integrin</t> αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.
    Apc Conjugated Antibody Against Integrin β6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/%CE%B26+integrin/pmc12804938-377-40-46?v=R%26D+Systems
    Average 92 stars, based on 10 article reviews
    apc conjugated antibody against integrin β6 - by Bioz Stars, 2026-07
    92/100 stars

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    1) Product Images from "Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection"

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    Journal: Nature Communications

    doi: 10.1038/s41467-025-67236-z

    a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.
    Figure Legend Snippet: a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

    Techniques Used: Transformation Assay, Infection, Software, Expressing, Staining, Flow Cytometry, Incubation

    a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

    Techniques Used: Western Blot, Expressing, Transduction, Control, Staining, Stable Transfection, Flow Cytometry, Infection, Incubation, Comparison

    a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.
    Figure Legend Snippet: a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

    Techniques Used: Expressing, Transduction, Staining, Flow Cytometry, Infection

    a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.
    Figure Legend Snippet: a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

    Techniques Used: Pull Down Assay, Sequencing, Negative Control, Magnetic Beads, Incubation, Western Blot, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Inhibition, Recombinant, Infection, Comparison

    a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Staining, Flow Cytometry, Incubation, Binding Assay, Quantitative RT-PCR, Virus, Infection, Comparison

    a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Infection, Expressing, Virus, Negative Control, Western Blot, Blocking Assay, Incubation, Software, Mutagenesis, Sequencing, Staining, Produced, Transfection, Comparison

    Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.
    Figure Legend Snippet: Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

    Techniques Used: Infection, Binding Assay



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    a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human <t>integrin</t> αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.
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    anti integrin β6  (Proteintech)
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    a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human <t>integrin</t> αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.
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    integrin β6  (Cell Signaling Technology Inc)
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    For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway <t>(Integrin</t> b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.
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    integrin β6 gfp  (Addgene inc)
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    For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway <t>(Integrin</t> b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.
    Integrin β6 Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fitc-labeled cytoplasmic tails of wildtype integrin β6, β1 and β1y795a  (Sangon Biotech)
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    Sangon Biotech fitc-labeled cytoplasmic tails of wildtype integrin β6, β1 and β1y795a
    For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway <t>(Integrin</t> b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.
    Fitc Labeled Cytoplasmic Tails Of Wildtype Integrin β6, β1 And β1y795a, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway <t>(Integrin</t> b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.
    Cytoplasmic Tail Peptides, Including Wildtype Integrin β6 And β1, As Well As Integrin Mutant β1y795a, supplied by GL Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a The graph displays the −log10-transformed RRA scores of genes enriched following infection with two SAFV-3 strains in HeLaN-∆SLC cells, analyzed using the MAGeCK software. The X -axis represents data from the screen using the SAFV-3 JPN08-356 strain, whereas the Y -axis shows results from the screen using the SAFV-3 JPN08-404 strain. The dotted line indicates the significance threshold of RRA = 0.01. Genes that met the criterion of RRA < 0.01 in both screens are highlighted in blue. The size of each dot reflects the combined enrichment across both screens, with larger dots indicating a greater sum of −log10 (RRA scores) from both experiments. b Expression of human integrin αV and integrin β8 in HeLaN-WT, HeLaN-∆AV, HeLaN-∆SLC∆AV, HeLaN-∆B8, and HeLaN-∆SLC∆B8 cells. The cells were stained with anti-integrin αV or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. c HeLaN-WT, HeLaN-∆AV, HeLaN-∆B8, HeLaN-∆SLC∆AV, and HeLaN-∆SLC∆B8 cells were infected with tenfold serial dilutions of SAFV-3 and viable cells were stained with crystal violet to assess infection levels. Images are representative of two independent experiments. d Multi-step growth kinetics of SAFV-3 in HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-WT cells. The cells were infected with SAFV-3 and incubated for up to 5 days. Data are presented as mean viral titers with s.d. ( n = 3). Statistical significance was determined using the two-sided Welch’s t -test. **, P < 0.01, *, P < 0.05, n.s. not significant. The dotted line indicates the limit of detection. Source data are provided as a Source Data file.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Transformation Assay, Infection, Software, Expressing, Staining, Flow Cytometry, Incubation

    a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a Western blot analysis of integrin αV (left panel) and integrin β8 (right panel) expression in BHK-21 cells. BHK-21 cells that were lentivirally transduced with either human integrin αV (BHK + human AV) or hamster integrin β8 (BHK + hamster B8) were used as positive controls. The anti-integrin αV antibody cross-reacted with both human and hamster integrin αV. Actin served as the loading control. b Expression of HS, integrin αV, and β8 in BHK-21 derivatives. BHK-21 cells were stained with anti-HS antibody (upper left panel). BHK-21 cells stably expressing human integrin αV and/or β8 (BHK + human AV, BHK + human B8, BHK + human AVB8), as well as the control cells, were stained with anti-integrin αV or anti-integrin αVβ8 antibodies. The cells were analyzed by flow cytometry. c Susceptibility analysis using SAF/UnaG in BHK-21 cells expressing human integrin αV and/or β8. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. The percentage of infected cells was determined by examining at least 1000 cells. Data are representative of two independent experiments. d One-step growth kinetics of SAFV-3 in BHK + human AV, BHK + human B8, BHK + human AVB8, and control cells. The cells were infected with SAFV-3 and incubated for up to 24 h. The dotted line indicates the limit of detection. e Western blot analysis of exogenous integrin β8 expression in BHK-21 cells lentivirally transduced with either mouse or hamster integrin β8. The anti-integrin β8 antibody cross-reacted with both mouse and hamster integrin β8. Actin served as the loading control. f Susceptibility analysis using SAF/UnaG in mouse and hamster integrin β8 expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and Hoechst-stained nuclei (blue, lower panel) were captured at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments. Data in ( c and d ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test ( c ) and the two-sided Welch’s t -test ( d ). **, P < 0.01, *, P < 0.05, n.s. not significant. Source data are provided as a Source Data file.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Western Blot, Expressing, Transduction, Control, Staining, Stable Transfection, Flow Cytometry, Infection, Incubation, Comparison

    a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a Expression of human integrin β subunits (β1, β3, β5, and β6) on the surface of BHK-21 derivatives. BHK-21 cells lentivirally transduced with the respective integrin β subunits were stained with the indicated antibodies and analyzed using flow cytometry. b Susceptibility analysis using SAF/UnaG in human integrin αV and the indicated β subunit expressing BHK-21 cells. UnaG-positive cells (green, upper panel) and nuclei stained with Hoechst (blue, lower panel) were imaged at 16 h post-infection. Scale bar, 200 μm. Images are representative of two independent experiments.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Expressing, Transduction, Staining, Flow Cytometry, Infection

    a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a Pull-down assay of SAFV-3 using heparin (left panel) and integrin αVβ8 (right panel). Heparin and Fc chimera of extracellular domains of integrin αVβ8, αVβ3, or the signal sequence (ss) of integrin αV (negative control) were prepared as complexes with magnetic beads. These complexes were incubated with SAFV-3, followed by western blot analysis of the bound virus using anti-SAFV-3 antiserum (left and right upper panels). The bottom right panel shows an image of the integrin-Fc complex on magnetic beads used for pulldown, detected using an anti-mouse IgG antibody. b Cell surface attachment assay for SAFV-3. HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 were incubated with SAFV-3, followed by RT-qPCR analysis of the bound virus. c Expression of human integrin β8 in HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells. To compare the expression levels of integrin αVβ8 on the cell surface, HeLaN-∆SLC and HeLaN-∆SLC + human AVB8 cells were stained with anti-integrin αVβ8 antibodies and analyzed by flow cytometry. d , e Inhibition of SAFV-3 attachment to the cell surface by soluble heparin ( d ) or recombinant integrin αVβ8 ( e ). HeLaN-WT cells ( d ) or HeLaN-∆SLC + human AVB8 cells ( e ) were incubated with SAFV-3 pretreated with 1 or 10 μg of soluble heparin or recombinant integrin αVβ8, respectively. Recombinant integrin αVβ3 was the negative control. After incubation at 4 °C for 2 h, bound virus was analyzed using RT-qPCR. f HeLaN-WT, HeLaN-∆SLC, HeLaN-∆B8, HeLaN-∆SLC∆B8, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of SAFV-3, and viable cells were stained with crystal violet to assess infection levels. All data are representative of two independent experiments. Data in ( b , d , and e ) represent means with s.d. ( n = 3). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Asterisks directly placed on bars indicate statistically significant differences compared to WT or untreated samples, while asterisks placed on the lines connecting bars denote statistically significant differences between those bars. Source data are provided as a Source Data file. Ag antigen, Fc fragment crystallizable region.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Pull Down Assay, Sequencing, Negative Control, Magnetic Beads, Incubation, Western Blot, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Inhibition, Recombinant, Infection, Comparison

    a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a Expression of HS and human integrin β8 in BHK-WT, BHK-∆SLC, BHK + human AVB8, BHK-∆SLC + human AVB8, and revertant cells expressing human SLC35B2 (BHK-∆SLC + human AVB8 + SLC). The cells were stained with anti-HS or anti-integrin αVβ8 antibodies and analyzed by flow cytometry. b Cell surface attachment assay for SAFV-3. BHK-WT and BHK-∆SLC cells were incubated with SAFV-3 at 4 °C for 2 h. Viral binding to HS was assessed by RT-qPCR quantification of cell-bound virus ( n = 3). c Susceptibility analysis using SAF/UnaG in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells. The cells used in this experiment were sorted to equalize the surface expression levels of integrin αVβ8 between BHK + human AVB8 and BHK-∆SLC + human AVB8 cells. UnaG-positive cells (green) and nuclei stained with Hoechst (blue) were imaged at 16 h post-infection. The percentage of infected cells was determined by examining at least 800 cells/well ( n = 4). Scale bar, 200 μm. d Cell surface attachment assay for SAFV-3 in BHK + human AVB8, BHK-∆SLC + human AVB8, and BHK-∆SLC + human AVB8 + SLC cells ( n = 3). Bar graphs in ( b– d ) are presented as means with s.d. Statistical significance was determined using the two-sided Welch’s t -test ( b ) and a one-way ANOVA with Dunnett’s multiple comparison test ( c , d ). **, P < 0.01, *, P < 0.05. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Expressing, Staining, Flow Cytometry, Incubation, Binding Assay, Quantitative RT-PCR, Virus, Infection, Comparison

    a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: a Viral infection analysis using integrin β8 mutants. BHK-21 cells expressing the human integrin β8 mutants (∆SDL, Y172N, and I208R) were inoculated with SAFV-3. After 2 days, virus titers were determined using the TCID 50 assay. Human integrin β3 was the negative control. The dotted line indicates the limit of detection. The right panel shows the results of western blot analysis of integrin β8 mutants and integrin β3 expression in BHK-21 cells. b Alignment of the amino acid sequences of puff A on VP2 of SAFV-3 (left panel) and CD loop I on VP1 of SAFV-2 (right panel). RGD-like sequences are highlighted. c Infection blocking assay using RGD peptide. Left panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRADS, or GRAES peptide at 4 °C for 30 min and then incubated with SAFV-3/UnaG virus for an additional 60 min. Right panel: HeLaN-∆SLC cells were pretreated with 10 or 100 μg of GRGDS, GRLDS, or GRAES peptide for 30 min and then incubated with SAFV-2/UnaG virus for an additional 60 min. GRGDS and GRAES peptides were positive and negative controls, respectively. The number of UnaG-positive cells at 14 h post-infection was counted using ImageJ software. d Schematic illustration of mutagenesis in RGD-like sequence. The mutated amino acid residues are highlighted. e HeLaN-∆SLC∆B8, HeLaN-∆SLC, and HeLaN-∆SLC + human AVB8 cells were infected with tenfold serial dilutions of mutant viruses carrying mutations in the RGD-like sequences, and viable cells were stained with crystal violet. Primary progeny virus produced from BHK cells transfected with infectious RNA (P-0 virus) was used. Bar graphs in ( a and c ) represent means with s.d. ( n = 3 in ( a ); n = 9 peptide-free and n = 3 peptide-added samples in ( c )). Statistical significance was determined using a one-way ANOVA with Dunnett’s multiple comparison test. **, P < 0.01, n.s. not significant. Statistical comparisons in ( c ) were made between peptide-free and peptide-added samples. All data are representative of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Infection, Expressing, Virus, Negative Control, Western Blot, Blocking Assay, Incubation, Software, Mutagenesis, Sequencing, Staining, Produced, Transfection, Comparison

    Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

    Journal: Nature Communications

    Article Title: Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection

    doi: 10.1038/s41467-025-67236-z

    Figure Lengend Snippet: Sulfated GAGs and integrin αVβ8 function as interconnected dual receptors for SAFV infection in HeLa-N cells. SAFV can directly bind to either sulfated GAGs or integrin αVβ8, while a portion of viruses bound to sulfated GAGs subsequently interact with integrin αVβ8. In addition, the data suggest the existence of a downstream molecule (factor X) required for an unspecified step in the viral entry process following sulfated GAGs binding.

    Article Snippet: For the detection of human integrin αV, β1, β3, β5, and β6, cells were stained with PE-conjugated antibodies against CD51 (integrin αV) (327910, BioLegend), CD29 (integrin β1) (303003, BioLegend), CD61 (integrin β3) (336405, BioLegend), integrin β5 (345203, BioLegend), and an APC-conjugated antibody against integrin β6 (FAB4155A, R&D Systems).

    Techniques: Infection, Binding Assay

    For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway (Integrin b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.

    Journal: bioRxiv

    Article Title: A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

    doi: 10.1101/2025.07.11.661192

    Figure Lengend Snippet: For this summary, examples of staining were obtained from multiple co-cultures. (A) hBECs derived from organoids were co cultured in ALI as homotypic recombinants (both epithelial cells and fibroblasts were obtained from the same tissue state (histologically-normal) or as heterotypic recombinants (epithelial cells were obtained from histologically-normal tissue and fibroblasts from progressive stages of cancer) for 21 or 42 days. The morphology of the formed epithelium was assessed by H&E staining and differentiation status by immunostaining for bronchial epithelial markers. Induction of squamous metaplasia was assessed by H&E staining and differentiation was assessed by immunostaining for squamous epithelial markers (Involucrin and KRT14). Esophageal embryologic origin of lung squamous metaplasia reported in vivo , was confirmed by immunostaining of in vitro cocultures for the esophagus-specific marker GBP6. Markers for dysplasia (TPX2 and aneuploidy) and for mechanotransdcution pathway (Integrin b6 and nuclear translocation of YAP) were also assessed by immunostaining in cocultures. (B) hBECs were co-cultured with fibroblasts obtained from histologically normal tissue adjacent to cancer (matched Normal-Associated Fibroblasts; mNAFs) or from cancer lesions (Carcinoma-Associated Fibroblasts; CAFs) for 21 days. The morphology of the formed epithelium was assessed by H&E staining (top). Cocultured fibroblasts were assessed for expression of fibroblast markers; PDGFRα was expressed in both mNAFs and CAFs, while both lacked the expression of the myofibroblast-specific marker alpha-smooth muscle actin (SMA) and pericyte markers RGS5 and PDGFRb (C) The expression of the squamous epithelial marker (Involucrin) in hBECs and of the fibroblast marker (Hsp47) was assessed by immunostaining in cocultures. (D) The fibroblast-generated ECM network was assessed by collagen staining. Interactions of hBECs with fibroblasts through ECM proteins in double-sided co-culture were visualized using overexposed Collagen I or Collagen IV staining, with arrows indicating collagen fibrils crossing the transwell pores and interacting with epithelial cells. (E) The percentage of each cell lineage in cocultures was quantified based on immunofluorescence staining. (F) The coherence of collagen filament alignment was quantified in collagen stained specimens in (B) using OrientationJ in ImageJ. (G) Schematic images showing serial sections across the entire Transwell membranes (6.5 mm diameter) after 21 days (left) or 42 days of co-culture (right). Vertical dashed lines represent the sections used for H&E staining. The presence of squamous metaplasia is indicated by red dashed lines. The overall percentage of surface areas with evidence of squamous tissue is shown.

    Article Snippet: The primary antibodies and dilutions used for immunofluorescence staining were as follows: acetylated Tubulin (Santa Cruz Biotechnologies (SCBT), sc23950, 1:100), p63 (Cell Signaling, 13109, 1:300) CC10 (SCBT, sc365992, 1:100), KRT5 (Abcam, ab64081, 1:500), Muc5AC (Abcam, ab198294, 1:250), Ki67 (Dako, M7240, 1:200), Involucrin (Abcam, ab68, 1:100), YAP (Cell Signaling, 140743, 1:200), Integrin β6 (Cell Signaling, 9513, 1:200) KRT14 (Biocare, CM185C, 1:100), Collagen I (Abcam, 1384921, 1:1500), p53 (Cell signaling, 488183, 1:100) SPC (Abcam, ab90716, 1:100), Collage IV (Abcam, 6586, 1:400), ZO-1 (Invitrogen, 33-9100 1:500), HSP47 (Abcam, 109117, 1:400), GPB6 (Sigma, HPA027744, 1:500), KRT10 (Abcam, ab9026, 1:100), PDGFRα (Cell signaling, 3164 1:100), alpha-Smooth Muscle Actin (Dako, 0851, 1:400), 53BP1(Cell Signaling, 88439, 1:200), p21 (Cell Signaling, 2947, 1:200), (TPX2 (Sigma, HPA005487, 1:200) and HPV16 E6 antigen (SCBT, SC460, 1:100)

    Techniques: Staining, Derivative Assay, Cell Culture, Immunostaining, In Vivo, In Vitro, Marker, Translocation Assay, Expressing, Generated, Co-Culture Assay, Immunofluorescence