Six-well tissue culture plates were coated using 50 mg/L purified human FN (Sigma Chemical, St. Louis, MO, United States) overnight at 4°C, and then treated with 3% bovine serum albumin for 3 h to prevent non-specific adhesion (Xiong et al., 2009 (link)). The human microvascular endothelial cell line (HMEC-1) was cultured as previously described (Seidl et al., 2012 (link)). Logarithmic-phase MRSA cells were added to FN-coated plates (5 × 103 cfu/mL) and endothelial cell monolayer-coated plates (5 × 105 cfu/mL; MOI = 1:1), and then incubated for 1 h at 37°C under static conditions. For FN binding assay, unbound bacteria were removed by washing the plates with PBS, and melted tryptic soy agar (TSA; 2 mL) was added into each well and allowed to solidify. For endothelial cell binding assay, unbound bacteria were removed by washing the plates with Hanks balanced salt solution (HBSS) and permeabilized using 1.0% Triton X-100 (Seidl et al., 2012 (link)), after which bacterial numbers per well were determined by serial dilutions and plating on TSA. Adherence was expressed as the percentage of the initial inoculum bound.
Human fn
Manufactured by Merck Group
Sourced in United States
Human FN is a lab equipment product manufactured by Merck Group. It is designed for the detection and measurement of fibronectin, a glycoprotein found in the extracellular matrix of various cell types. The core function of this product is to facilitate the quantification and analysis of fibronectin levels in biological samples.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Trifluoroacetic acid, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
3 aminopropyltriethoxysilane aptes, by Thermo Fisher Scientific (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Mmp 2, by R&D Systems (1 mentions)
Rat collagen type 1, by Merck Group (1 mentions)
Dq gelatin, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg antibody, by Boster Bio (1 mentions)
Pierce biotin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Merck Group (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Cytiva (1 mentions)
Ve cadherin clone 55 7h1, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Kindlin 2, by Merck Group (1 mentions)
Vegfr2, by R&D Systems (1 mentions)
Puromycin, by Merck Group (1 mentions)
7 protocols using human fn
1
MRSA Adhesion to FN and Endothelial Cells
2
MALDI-TOF Mass Spectrometry Workflow
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Bovine gelatin (G9382, type B) and human FN were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin (sequencing grade) was obtained from Promega (Madison, WI, USA). Trifluoroacetic acid (TFA) and acetonitrile (ACN) were purchased from Fisher Scientific (Fair Lawn, JN, USA). α-Cyano-4-hydroxycinnamic acid (CHCA) was purchased from Bruker (Billerica, MA, USA). 3-Aminopropyltriethoxysilane (APTES) was purchased from Alfa Aesar (Haverhill, MA, USA). An unmodified Anachip was purchased from Farfield (Sensors Ltd., Salford, UK). The four synthesized peptides were purified by Beijing Scilight Biotechnology Led. Co. (Beijing, China).
3
Characterization of Synthetic Peptides
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R1R2 and the scrambled peptide were custom-synthesized by Kelowna International Scientific Inc. (Taipei, Taiwan). The sequence of R1R2 used in this study was GLNGENQKEPEQGERGEAGPPLSGLSGNNQGRPSLPGLNGENQKEPEQGERGEAGPP , and that of the scrambled peptide was LGNEGQKNPEREEGQPAGEGLGLSPQNSGNLPRSGGNGLPQEKENEGPEQPERPAGG . For competitive enzyme-linked immunosorbent assay (ELISA), human FN and rat type I collagen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse CXCL12 (SDF-1 beta, BioLegend, San Diego, CA, USA), human CXCL12 (SDF-1 beta, R&D, Minneapolis, MN, USA), human and mouse active MMP-9 (Abcam, Cambridge, MA, USA) and MMP-2 (R&D, Minneapolis, MN, USA) proteins, and DQ Gelatin® (Invitrogen, Waltham, MA, USA) were used for in vitro assays.
4
Recombinant Protein Binding Assay
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Binding studies were performed by Far-Western blotting. Recombinant proteins were separated by SDS-PAGE, then electroblotted onto PVDF membrane and blocked with 5% (w/v) skimmed milk at 4°C overnight. Subsequently, the membrane was incubated with human LN (Sigma; 5 μg/ml) or human FN (Sigma; 10 μg/ml) for 24 h at 4°C. Frequently, the membranes were washed three times with TBST and incubated with rabbit anti-LN (Abcam; 1:2000 dilution) or rabbit anti-FN (Boster; 1:2000 dilution) antibody at 37°C for 2 h, rinsed three times with TBST and then incubated with goat anti-rabbit IgG antibody (Boster; 1:2000 dilution). The specific bands were developed using substrate solution 3,3′-diaminobenzidine (DAB; Tiangen, China). The recombinant protein MRP was performed as negative control.
5
Biotinylation of Fibronectin and Campylobacter Binding
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Human FN (Sigma-Aldrich) was suspended in 1 mL phosphate buffered saline (PBS) to a concentration of 5 µM, and biotinylated by adding 1 mg of Sulfo-NHS-LC-Biotin for 3 h at room temperature. Excess biotin was removed using a Zeba desalting column (40k MWCO, Thermo Fisher Scientific, Waltham, MA, USA) and dialyzing overnight in PBS (40k MWCO) at 4 °C. The C. jejuni isolates were grown overnight in MH broth, pelleted, and resuspended to an OD540 = 0.3 in PBS (~3 × 108 CFU/mL). A suspension containing 80 µL of bacteria in PBS and 20 µL of biotinylated FN was mixed, incubated for 1 h at room temperature, and C. jejuni pelleted by centrifugation. The amount of biotinylated FN in the supernatant was determined using a Pierce Biotin Quantitation Kit, as outlined by the manufacturer (Thermo Fisher Scientific).
6
Integrin β3 Signaling Pathway
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Antibodies used were directed against β-actin (clone C4; Chemicon), integrin β3 (clone C-17 from Dr. E. van der Schoot, Sanquin Research, Amsterdam, The Netherlands; integrin β1 (clone TS2/16, Developmental Studies Hybridoma Bank), kindlin-2 (from Dr. R. Faessler, Max Planck Institute of Biochemistry, Martinsried, Germany), P(Y) (clone 4G10 from Sigma-Aldrich), Rac (Transduction laboratories), RhoA (Cell Signaling), VE-Cadherin (clone 55-7H1, BD biosciences) and VEGFR2 (R&D Systems). The β3 deletion mutants were obtained from Dr. J. Ylanne (University of Jyvaskyla, Finland) and recloned into LZRS-IRES-zeo as described previously [92] , while Rac and RhoA mutants fused to mCherry or GFP were from Dr. J. Collard (Netherlands Cancer Institute, Amsterdam, The Netherlands). GFP-tensin-1 was from Dr. K. Yamada (National Institute of Health, Bethesda, MD), Rhotekin-RBD and PAK-CRIB peptides were home-made, human fibrinogen was from CSL Behring, collagen-coated Cytodex beads, puromycin, zeocin, human thrombin, and human FN were from Sigma-Aldrich. TRITC-, FITC-, and Cy5-conjugated secondary antibodies, phalloidins, and DAPI were from Molecular Probes, Fugene was from Promega, and HRPconjugated secondary antibodies were from Amersham.
7
Protein Adsorption on Surfaces
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Protein solutions. Bovine serum albumin (BSA; Sigma-Aldrich), human Fbg (Merck Millipore), human Fn (Sigma-Aldrich) and Col I (Sigma-Aldrich) were used at different concentrations established to match human blood plasma ratios. BSA was used at 5 mg/mL in a phosphatebuffered saline solution (PBS; Sigma), Fbg at 500 µg/mL in PBS, Fn at 50 µg/mL in PBS and Col I at 10 µg/mL in acetate buffer (0.1 M, pH 5.6).
Adsorption from monoprotein solutions. A QCM-D system (Q-sense Model E4, Biolin Scientific, Sweden) was used to monitor protein adsorption study by measuring the frequency and dissipation values during the process. Experiments were conducted at 37 °C and with a constant flow rate of 25 µL/min. After a stable baseline was reached with PBS, each of the protein solution was individually introduced into the QCM-D module, and the flow was maintained until reaching saturation. Then, PBS was again introduced into the chamber to remove the reversibly adsorbed proteins on the surface and to obtain a new plateau. QCM-D data were analyzed using the Q-Tools Software using the Voight model. [39, 40]
Adsorption from monoprotein solutions. A QCM-D system (Q-sense Model E4, Biolin Scientific, Sweden) was used to monitor protein adsorption study by measuring the frequency and dissipation values during the process. Experiments were conducted at 37 °C and with a constant flow rate of 25 µL/min. After a stable baseline was reached with PBS, each of the protein solution was individually introduced into the QCM-D module, and the flow was maintained until reaching saturation. Then, PBS was again introduced into the chamber to remove the reversibly adsorbed proteins on the surface and to obtain a new plateau. QCM-D data were analyzed using the Q-Tools Software using the Voight model. [39, 40]
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