Journal: Communications Chemistry

Article Title: Paper-based electrochemical device for early detection of integrin αvβ6 expressing tumors

doi: 10.1038/s42004-024-01144-z

Figure Lengend Snippet: a Engineering of the paper-based electrochemical strip following the screen-printing of conductive ink, modification with a dispersion of gold nanoparticles, covalent engineering of the αvβ6-selective probe and saturation of the surface with mercaptohexanol (6-MCH); b Impedimetric measurements show an increase of the semi-circle (charge transfer resistance, Rct) when the binding between probe and protein occurs (orange line) in comparison with absence of target (black line).

Article Snippet: 25 µg of proteins lysates from cells grown in serum starved condition for 24 h and S-EVs (collected as described in “S-EVs isolation” section) were analysed by SDS-PAGE and Western Blot (WB) performed with antibodies: αvβ6 integrin (1:500 Bioss, bs-5791R), TSG101 (1:500, abcam cat. ab30871), CD81 (1:500, cell signaling #56039), β-actin (1:500, ab30871), γ-tubulin (1:500, sc-17787) Enhanced chemiluminescence (ECL) immunodetection reagents were from GE Healthcare.

Techniques: Stripping Membranes, Modification, Dispersion, Binding Assay, Comparison

Journal: Communications Chemistry

Article Title: Paper-based electrochemical device for early detection of integrin αvβ6 expressing tumors

doi: 10.1038/s42004-024-01144-z

Figure Lengend Snippet: a Optimization of the probe length to be immobilized onto the strip. For each experiment each probe at 100 nM concentration has been immobilized onto the strip; the impedimetric measurements have been carried out in presence of 5 ng/mL αvβ6. b Nyquist plots for the nanoengineered biosensor 3 challenged in absence (black dots) and in presence of different concentration of αvβ6, i.e., 1 (cyan dots), 2 (gray dots), 4 (orange dots), 5 (red dots), 10 (green dots) and 20 ng/mL (blue dots). The measurements were carried out as follows: the nanoengineered biosensor was covered with a solution containing the chosen level of αvβ6. After 30 min at room temperature, the biosensors were washed with phosphate buffer, and then covered with a solution containing 1 mM [Fe(CN) 6 ] 3−/4− dissolved in 0.1 M KCl. A potential of 0.2 V and a AC amplitude of 10 mV in a frequency range of 100 kHz to 0.1 Hz have been utilized. The X and Y axes represent, respectively, the real and imaginary components of impedance. The inset i shows the Randles equivalent electrical circuit that has been used to fit the spectra, comprising the electrolyte resistance, Re, in series with a parallel combination of Rct (charge transfer resistance), Zw (diffusion of the analytes in solution and corresponding to Warburg impedance straight line of the curves) and CPE (Constant Phase Element). c Linear range comprised between 1 and 20 ng/mL αvβ6. Inset shows the complete semi-log correlation in a wider range of αvβ6 concentrations, namely 0.01–50 ng/mL.

Article Snippet: 25 µg of proteins lysates from cells grown in serum starved condition for 24 h and S-EVs (collected as described in “S-EVs isolation” section) were analysed by SDS-PAGE and Western Blot (WB) performed with antibodies: αvβ6 integrin (1:500 Bioss, bs-5791R), TSG101 (1:500, abcam cat. ab30871), CD81 (1:500, cell signaling #56039), β-actin (1:500, ab30871), γ-tubulin (1:500, sc-17787) Enhanced chemiluminescence (ECL) immunodetection reagents were from GE Healthcare.

Techniques: Stripping Membranes, Concentration Assay, Diffusion-based Assay

Journal: Communications Chemistry

Article Title: Paper-based electrochemical device for early detection of integrin αvβ6 expressing tumors

doi: 10.1038/s42004-024-01144-z

Figure Lengend Snippet: a In presence of 2 ng/mL and ( b ) in presence of 20 ng/mL of αvβ6 (gray bars), α5β1 (green bars) and αvβ3 (orange bars) integrins. The bars are the results of 5 replicates. The dashed red lines indicate the signal observed in absence of targets. The concentrations of integrins have been selected in agreement with the linearity range of the platform.

Article Snippet: 25 µg of proteins lysates from cells grown in serum starved condition for 24 h and S-EVs (collected as described in “S-EVs isolation” section) were analysed by SDS-PAGE and Western Blot (WB) performed with antibodies: αvβ6 integrin (1:500 Bioss, bs-5791R), TSG101 (1:500, abcam cat. ab30871), CD81 (1:500, cell signaling #56039), β-actin (1:500, ab30871), γ-tubulin (1:500, sc-17787) Enhanced chemiluminescence (ECL) immunodetection reagents were from GE Healthcare.

Techniques:

Journal: Communications Chemistry

Article Title: Paper-based electrochemical device for early detection of integrin αvβ6 expressing tumors

doi: 10.1038/s42004-024-01144-z

Figure Lengend Snippet: a Representative image of TRPS measurements showing particle size and concentration of S-EVs derived from DU145R80 (R80) cancer cell line (see text). b Upper panels: representative images of Transmission Electron micrograph (TEM) of S-EVs isolated from cell culture media. Magnification 49000X, scale bar 200 nm. The morphology is observed by negative staining. Lower panels: representative images of Immuno Electron micrograph (IEM) of S-EVs, labeled using antibodies specific to αvβ6 integrin and antibody binding was confirmed by Protein A gold-conjugate to 10 nm gold particles. Magnification 120000X, scale bars 100 nm. c Integrin β6 expression by ELISA expressed in terms of fold changes of S-EVs from PC3, A549 and HCT116 compared to R80-derived S-EVs. d Western blot analysis of S-EVs lysates from R80, PC3, HCT116 and A549 and tested with antibodies as indicated in the figure. Both TSG101 and CD81 were tested as positive EVs markers. β actin served as negative control. e Densitometry of Integrin αVβ6/CD81 expression in EVs, expressed as fold changes in either HEK293- or H460- S-EVs compared to PC3-derived S-EVs. Inset shows Western blot analysis of S-EVs lysates from PC3, HEK293 and H460 tested with antibodies as indicated in the figure. Both TSG101 and CD81 were tested as positive EVs markers. ɤ-tubulin served as negative control .

Article Snippet: 25 µg of proteins lysates from cells grown in serum starved condition for 24 h and S-EVs (collected as described in “S-EVs isolation” section) were analysed by SDS-PAGE and Western Blot (WB) performed with antibodies: αvβ6 integrin (1:500 Bioss, bs-5791R), TSG101 (1:500, abcam cat. ab30871), CD81 (1:500, cell signaling #56039), β-actin (1:500, ab30871), γ-tubulin (1:500, sc-17787) Enhanced chemiluminescence (ECL) immunodetection reagents were from GE Healthcare.

Techniques: Concentration Assay, Derivative Assay, Transmission Assay, Isolation, Cell Culture, Negative Staining, Labeling, Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue

Journal: Bone Research

Article Title: Mechanosignaling activation of TGFβ maintains intervertebral disc homeostasis

doi: 10.1038/boneres.2017.8

Figure Lengend Snippet: Integrin α V β 6 induces TGFβ activation in response to mechanical stress. ( a – h ) Representative images and quantification of immunostaining of IVD sections with antibodies against ( a , b ) α V β 6 , ( c , d ) β8, ( e , f ) α V β 5 , and ( g , h ) α V β 3 (brown) in LSI and sham mice. Hematoxylin stains nuclei purple. n =6 per group. Data are shown as mean±s.d. * P <0.05, ** P <0.01 (two-sided Student’s t test). ( i ) Immunostaining for pSmad2/3 in the NC cells (brown). Hematoxylin stains nuclei purple. ( j ) Safranin O staining of IVD sections from an IVD ex vivo compression model with application of either Veh (rows 1 and 3), recombinant mouse TGFβ1 (row 2), RGD peptide, TGFβ or α V β 6 neutralizing antibodies (bottom 3 rows). ( k ) Quantification of pSmad2/3 + cells in i . ( l ) Western blot analysis of pSmad2 and total Smad2 levels in the IVD. n =6 per group in histological examination. Representative image from three independent experiments were conducted for ( l ). Data are shown as mean±s.d. * P <0.05, ** P <0.01 (ANOVA).

Article Snippet: Sections were incubated with primary antibodies to mouse aggrecan (1:200, AB1031, Millipore, Billerica, MA, USA), CCN2 (1:400, ab6992, Abcam), pSmad2/3 (1:200, sc-11769, Santa Cruz, Dallas, TX, USA), integrin α V β 6 (1:100, bs-5791R-Biotin, Bioss, Woburn, MA, USA), integrin α V β 3 (1:100, bs-1310R, Bioss), integrin α V β 5 (1:100, bs-1356R, Bioss), integrin β 8 (1:300, ab80673, Abcam), integrin α V (Santa Cruz, 1:100, sc-6617-R), integrin β 6 (Santa Cruz, 1:100, sc-6632), and TGFβRII (1:100, sc-400, Santa Cruz) at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Staining, Ex Vivo, Recombinant, Western Blot

Journal: Bone Research

Article Title: Mechanosignaling activation of TGFβ maintains intervertebral disc homeostasis

doi: 10.1038/boneres.2017.8

Figure Lengend Snippet: Conditional knockout of integrin α v impedes functional transition of NC cells. ( a , b ) Immunostaining of IVD sections from α v −/− mice and their wild-type α v +/+ littermates (4-week-old) with antibodies against α v ( a ) and β 6 ( b ) (brown). Hematoxylin stains nuclei purple. ( c , d ) Immunostaining for pSmad2/3 ( c ) and α V β 6 ( d ) in NC cells. ( e , f ) Immunostaining for CCN2 ( e ) and Acan ( f ) in NC cells. ( g , h ) Quantification of pSmad2/3 ( c ) and α V β 6 ( d ) expression in NC cells. ( i , j ) Quantification of CCN2 ( i ) and α V β 6 ( j ) expression in NC cells. ( k ) Western blot analysis of α v level in NC cells. ( l ) Western blot analysis of pSmad2 and Smad2 levels in NC cells. ( m ) Safranin O-fast green staining of L 3–4 IVDs showing enlarged vacuoles with less proteoglycans (red orange) in α v −/− mice relative to their α v +/+ littermates (4-week-old). ( n ) Safranin O staining images of the IVDs from sham-operated or LSI TgfbrII −/− mice and α v −/− mice (8-week-old) at 2 weeks post surgery. n =3 per group. Data are shown as mean±s.d. * P <0.05, ** P <0.01 (two-sided Student’s t test).

Article Snippet: Sections were incubated with primary antibodies to mouse aggrecan (1:200, AB1031, Millipore, Billerica, MA, USA), CCN2 (1:400, ab6992, Abcam), pSmad2/3 (1:200, sc-11769, Santa Cruz, Dallas, TX, USA), integrin α V β 6 (1:100, bs-5791R-Biotin, Bioss, Woburn, MA, USA), integrin α V β 3 (1:100, bs-1310R, Bioss), integrin α V β 5 (1:100, bs-1356R, Bioss), integrin β 8 (1:300, ab80673, Abcam), integrin α V (Santa Cruz, 1:100, sc-6617-R), integrin β 6 (Santa Cruz, 1:100, sc-6632), and TGFβRII (1:100, sc-400, Santa Cruz) at 4 °C overnight.

Techniques: Knock-Out, Functional Assay, Immunostaining, Expressing, Western Blot, Staining

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Binding Assay, Expressing

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Overview of αvβ6 complexed with the 20mer A20M3 peptide. αv (light green), β6 (pink) and A20M3 (grey) chains are shown as cartoon traces. Side chains of the RGD motif are shown as sticks and spheres. (B) Superposition of αvβ6 in surface representation in complex with A20M1 (yellow carbons), A20M3 (grey carbons) and A20FMDV2 (salmon carbons). The position of manganese ions (cyan spheres) shown superimposed were derived from the human αvβ6/TGFβ1 complex (PDB ID: 5ffo). Labeled aa refer to the A20M3 sequence. (C) Superposition of the αvβ6/A20M3 complex onto the αvβ8 structure (blue carbons). Note that the A20M3 peptide is not shown here. Residue numbering refers to the Uniprot entry Q9Z0T9 (mouse integrin subunit β6) and the A20M3 peptide sequence (Arg16).

Article Snippet: The A20M1 and A20M3 consist of aa residues 490–509 of the M1 fiber (Uniprot P19721), and 452–471 of the M3 fiber (GenBank: ACJ14524.1) sequence, respectively. sITGs were obtained from R&D systems (Bio-techne brand, USA), including mouse and human αvβ3 (7889-AV-050/3050-AV-050), αvβ5 (7706-AV-050/2528-AV-050), αvβ6 (7480-AV-050/3817-AV-050), αvβ8 (8314-AV-050/4135-AV-050), recombinantly expressed in and secreted from CHO cells (quality control shown in ).

Techniques: Derivative Assay, Labeling, Sequencing, Residue

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Clinical quantification of the integrin αvβ6 by [ 18 F]FB-A20FMDV2 positron emission tomography in healthy and fibrotic human lung (PETAL Study)

doi: 10.1007/s00259-019-04586-z

Figure Lengend Snippet: Objectives and endpoints

Article Snippet: A number of drugs targeting αvβ6 integrin across fibrotic diseases are currently in development including an inhaled small molecule inhibitor of αvβ6 under clinical development by GlaxoSmithKline R&D and an intravenous monoclonal antibody under development by Biogen (see studies registered on clinical trials.gov : NCT01371305, NCT02612051 and NCT03069989) [ ].

Techniques: Computed Tomography