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: 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