Epithelial cells, grown in 96-well plates, were incubated for 30 min with 100 μL/well of DMEM-F12 only or supplemented with fibronectin (1 μg/well), antibodies anti-integrin α5β1 that block the interaction with RGD site in fibronectin (Abcam) or anti-integrin α5 (Santa Cruz Biotechnology). Then, medium was aspirated and bacteria were added to the monolayer (in triplicate), with a multiplicity of infection (MOI) of 10. The plates were incubated at 37°C in 5% CO2 for 3 h and then washed three times with PBS. Cells were lysed with a solution containing 0.1% (v/v) Triton X-100/PBS and serial dilutions of the lysates were plated on LB agar. The number of adherent bacteria was determined by counting colonies forming units (CFU).
Fibronectin
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Fibronectin is a high molecular weight glycoprotein found in the extracellular matrix and plasma. It plays a crucial role in cell adhesion, growth, migration, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Abcam (54 mentions)
Vimentin, by Abcam (47 mentions)
E cadherin, by Abcam (45 mentions)
Collagen 1, by Abcam (38 mentions)
Gapdh, by Abcam (30 mentions)
Collagen 4, by Abcam (28 mentions)
N cadherin, by Abcam (24 mentions)
Gapdh, by Cell Signaling Technology (22 mentions)
β actin, by Abcam (20 mentions)
Laminin, by Abcam (19 mentions)
β actin, by Merck Group (18 mentions)
Pvdf membrane, by Merck Group (16 mentions)
β actin, by Cell Signaling Technology (16 mentions)
E cadherin, by BD (15 mentions)
Tgf β1, by Abcam (15 mentions)
E cadherin, by Cell Signaling Technology (15 mentions)
Collagen 3, by Abcam (11 mentions)
Vimentin, by Cell Signaling Technology (11 mentions)
Type 1 collagen, by Abcam (10 mentions)
Snail, by Abcam (10 mentions)
324 protocols using fibronectin
1
Quantifying Bacterial Adhesion to Epithelial Cells
2
Western Blot Analysis of Fibrosis Markers
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After the hTECs were removed from culture dishes, the proteins were retrieved using radioimmunoprecipitation assay (RIPA) buffer containing Halt protease inhibitor (Pierce, Rockford, IL, USA). Western blotting was performed using primary antibodies against fibronectin (Abcam), CCL8 (Novus Biologics), E-cadherin (Abcam), BCL-2 (Santa Cruz Biotechnology, Dallas, TX, USA), CD44 (Abcam), CCR2 (Abcam), and β-actin (Sigma-Aldrich) CD44 (Abcam), a transmembrane receptor, interacts with cell-matrix components, such as fibronectin and collagen, and is a potential marker of fibrosis. Equal amounts of extracted proteins (20~40 μg) were isolated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto Immobilon-FL 0.4 μm of polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) and anti-mouse IgG (Cell Signaling Technology) were used as horseradish peroxidase-conjugated secondary antibodies. The immunoblot bands were visualized and images were captured using an ImageQuant LAS 4000 Mini instrument (GE Healthcare, Princeton, NJ, USA). Western blotting results were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
3
Characterization of Cell Morphology and Surface Proteins
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The cell morphology and surface protein expression of the two types of cells were compared. Morphologic observations of the third passage dADSCs and VFFs were made using an inverted microscope. The cell surface protein expression levels were identified by cell immunofluorescence.
When the cells reached 80% to 90% confluence, they were fixed in ice-cold 4% paraformaldehyde for 10 minutes and rinsed with phosphate-buffered saline (PBS). They were permeabilized in PBS containing 0.3% Triton X-100 for 30 minutes at room temperature (RT). All cells were blocked (3% horse serum in PBS, 30 minutes, RT) and incubated for 2 hours (4°C) with the following primary antibodies: vimentin (monoclonal mouse vimentin, 1∶200; Novus, Littleton, CO, USA) and fibronectin (polyclonal rabbit fibronectin, 1∶100; Abcam, Cambridge, MA, USA). After a thorough washing, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1∶400; Molecular Probes, Eugene, OR, USA) and 594-conjugated goat anti-mouse IgG (1∶400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour (RT). All slides were counterstained using Hoechst 33342 (1∶1000; Roche; 10 minutes, RT) for nuclear staining.
When the cells reached 80% to 90% confluence, they were fixed in ice-cold 4% paraformaldehyde for 10 minutes and rinsed with phosphate-buffered saline (PBS). They were permeabilized in PBS containing 0.3% Triton X-100 for 30 minutes at room temperature (RT). All cells were blocked (3% horse serum in PBS, 30 minutes, RT) and incubated for 2 hours (4°C) with the following primary antibodies: vimentin (monoclonal mouse vimentin, 1∶200; Novus, Littleton, CO, USA) and fibronectin (polyclonal rabbit fibronectin, 1∶100; Abcam, Cambridge, MA, USA). After a thorough washing, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1∶400; Molecular Probes, Eugene, OR, USA) and 594-conjugated goat anti-mouse IgG (1∶400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour (RT). All slides were counterstained using Hoechst 33342 (1∶1000; Roche; 10 minutes, RT) for nuclear staining.
4
Characterization of ECM Protein Expression
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All histological samples were fixed in 10% v/v buffered neutral formalin and stained with haematoxylin and eosin (H+E) for light microscopy.
IHC was performed to assess retention of key ECM and basement membrane proteins collagens I and IV, laminin and fibronectin (all Abcam/UK). All samples were treated identically with regards to fixation, antigen retrieval, antibody staining, blocking and development. Briefly, paraffin-embedded sections were dewaxed and blocked with 1% v/v hydrogen peroxide in methanol for 10 minutes. Heat-mediated antigen retrieval was performed in 0.01M sodium citrate buffer (Sigma/UK), pH 6.0, followed by Avidin-Biotin blocking (Dako/UK). Sections were blocked for 1 hour with 5% v/v goat serum in PBS, and primary antibodies were incubated overnight at 4 o C.
Primary antibody dilutions in blocking solution (5% v/v goat serum in PBS) were: collagen I 1:250, collagen IV 1:500, laminin 1:50, and fibronectin 1:500 (Abcam/UK).
After washing sections in 0.1% v/v TWEEN (Sigma/UK) in PBS, pH 7.4, hydrogen peroxidase conjugated secondary antibody (Abcam/UK) was applied at 1:100 for 1 h at room temperature then washed again in TWEEN solution. Slides were developed using 3,3'-diaminobenzidine (Dako/UK) until good staining intensity was observed.
Species immunoglobulin and PBS served as negative controls. Haematoxylin was used for nuclear counterstaining.
IHC was performed to assess retention of key ECM and basement membrane proteins collagens I and IV, laminin and fibronectin (all Abcam/UK). All samples were treated identically with regards to fixation, antigen retrieval, antibody staining, blocking and development. Briefly, paraffin-embedded sections were dewaxed and blocked with 1% v/v hydrogen peroxide in methanol for 10 minutes. Heat-mediated antigen retrieval was performed in 0.01M sodium citrate buffer (Sigma/UK), pH 6.0, followed by Avidin-Biotin blocking (Dako/UK). Sections were blocked for 1 hour with 5% v/v goat serum in PBS, and primary antibodies were incubated overnight at 4 o C.
Primary antibody dilutions in blocking solution (5% v/v goat serum in PBS) were: collagen I 1:250, collagen IV 1:500, laminin 1:50, and fibronectin 1:500 (Abcam/UK).
After washing sections in 0.1% v/v TWEEN (Sigma/UK) in PBS, pH 7.4, hydrogen peroxidase conjugated secondary antibody (Abcam/UK) was applied at 1:100 for 1 h at room temperature then washed again in TWEEN solution. Slides were developed using 3,3'-diaminobenzidine (Dako/UK) until good staining intensity was observed.
Species immunoglobulin and PBS served as negative controls. Haematoxylin was used for nuclear counterstaining.
5
Protein Extraction and Immunoblotting from Rat Kidneys
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Total tissue and cell protein extracted from rat kidneys and GMCs were lysed with RIPA lysis buffer (Beyotime, China) and measured with a BCA Protein Assay Kit (Beyotime, China). Nuclear protein was extracted using the Nuclear and Cytoplasmic Protein Extraction kit (cat. no. P0028; Beyotime Institute of Biotechnology, Nanjing, China) following the manufacturer’s instructions. The protein was separated by 10% SDS–PAGE and electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and incubated with the following primary antibodies overnight at 4 °C: TRPC6, collagen IV, collagen I, fibronectin, and NFAT2 from Abcam (Cambridge, MA, USA) as well as β-actin and histone 3 from ABclonal (Wuhan, China). On the next day, HRP-conjugated goat anti-rabbit or goat anti-mouse IgG, which served as the secondary antibody, was incubated with the membranes for 2 h at room temperature to detect immune response bands. ECL chemiluminescence was used for imaging.
6
Knockdown of MAGEC2 in Hepatocellular Carcinoma
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One HCC cell line (MHCC97H) was generously provided by Yuanduan Biological Co., Ltd. (Jiangsu, China). Two other HCC cell lines (SK-H-1 and HepG2) and the human liver cell line LO-2 were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). For MAGEC2 knockdown, the target sequence of the short hairpin RNA (shRNA) was as follows: 5ʹ- CCT CTT CCA CTT TGT ACT T -3ʹ (Gene ID: NM_016249). The shRNA targeting MAGEC2 (sh-MAGEC2) was cloned into pLKD-CMV-G&PR-U6-shRNA (Obio Technology, Co., Ltd., Shanghai, China). An unrelated random sequence was used as an sh-MAGEC2 negative control (sh-MAGEC2-NC). After viral transduction, MAGEC2 expression was evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB) analyses. The primary antibodies, including MAGEC2, E-cadherin, N-cadherin, fibronectin, Slug, cleaved caspase-3, Cyclin D1, Cyclin E and Ki67, were all purchased from Abcam Co., Ltd. (Abcam, Cambridge, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).
7
Protein Expression Analysis in Cells
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Cells were collected, washed, and extracted with PRO-PREP™ protein extraction solution (iNtRON Biotech, Sungnam, Korea). Fifty micrograms of proteins were separated on an 8% sodium dodecyl sulfate-polyacryamide gel by electrophoresis. The proteins were transferred to Nitrocellulose membranes (Bio-rad, Hercules, CA). The membranes were incubated with antibodies of Collagen type 1, Collagen type 4, Fibronectin, Elastin, TGFb Receptor1, SMAD2, p-SMAD2, SMAD3, p-SMAD3 and GAPDH (1:1000; abcam, Cambridge, USA). Then, the membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase (1:5000, abcam). Membranes were developed using ECL (GE Healthcare, Pittsburgh, USA) and quantified using a densitometer. Mean pixel density was quantified using Image J analysis Representative western blot of Collagen type 1, Collagen type 4, Fibronectin, Elastin, TGFb Receptor1, SMAD2, p-SMAD2, SMAD3 and p-SMAD3 those of either total protein or GAPDH to correct for protein loading in the case of cellular lysate extracts.
8
Immunofluorescence Imaging of Cell Markers
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For immunofluorescence staining, the cells were incubated with primary antibodies against VE-cadherin (Cell Signaling Technology [CST], USA), vimentin (CST) or fibronectin (Abcam), followed by incubation with Alexa Fluor 488- or 555-conjugated secondary antibodies (Invitrogen, USA). For confocal microscopy, the cells grown on coverslips were counterstained with DAPI and were imaged using a confocal laser-scanning microscope (Carl Zeiss, Germany) with a core data acquisition system (Applied Precision).
9
Immunofluorescence Assay for Cell-Cell Junctions
10
Immunohistochemical analysis of cancer markers
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After deparaffinization and rehydration, the paraffin-embedded sections were subjected to high pressure for 2 min for antigenic retrieval. The slides were incubated overnight at 4°C with the following primary antibodies: Hes1 (Bioss, dilution 1:500), PTEN (Cell Signaling, dilution 1:200), E-cadherin (BD, dilution 1:300), Fibronectin (Abcam, dilution 1:300), and Snail (Cell Signaling, clone C15D3, dilution 1:100). PBS were used as negative controls. The sections were then incubated with DAB for 2 min. The staining intensity of tumor cells was grouped into four grades: 0, no staining; 1, weak staining; 2, modest staining; and 3, strong staining. The positive staining ratio of tumor cells was classified into four grades: 0, no positive tumor cells; 1, < 10% positive tumor cells; 2, 10–50% positive tumor cells; and 3, > 50% positive tumor cells. The positive staining ratio of tumor cells = hes1-positive tumor cells/total tumor cellsX100%. The general IHC results were calculated by multiplying the positive staining grade by the intensity grade (0, 1, 2, 3, 4, 6, and 9). Finally, general IHC results ≦4 and ≧6 were defined as low and high expression, respectively. Two pathologists examined and scored IHC results blindly without knowing the clinical characteristics and prognosis.
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