Journal: bioRxiv

Article Title: A common polymorphism that protects from cardiovascular disease increases fibronectin processing and secretion

doi: 10.1101/2021.03.09.434522

Figure Lengend Snippet: Constructs encoding either variant of pFN1, tagged with a C-terminal Hemagglutinin tag, were transfected for 22 h in HEK293T or HuH-7 cells, as indicated A, Media and lysates were then analyzed by Western blot using an HA-specific antibody; no signal was detected in untransfected cells. Two independent experiments are shown for each construct. B, Quantification of FN1-HA Western blots. Signals from media (GFP) and cytosol (pRSV40 Renilla) were quantified for each transfection and data is expressed as the secreted to cellular signals for Q15 and L15 (± 95% C.I.). Each point represents a distinct experiment.

Article Snippet: The full-length pFN1 construct was obtained from Addgene (Fibronectin-human-plasma in pMAX; Plasmid #120401 ).

Techniques: Construct, Variant Assay, Transfection, Western Blot

Journal: bioRxiv

Article Title: CellWell: A micropatterned biphasic nanocomposite platform for culturing chondrocytes

doi: 10.1101/790030

Figure Lengend Snippet: Murine femoral cap hip explants (A) and primary human articular chondrocytes (B) and (C) were plated on fibronectin-coated coverglass using standard 2D cell culture techniques, fixed with 4% paraformaldehyde, stained with ActinRed 555 Ready probes® Reagent (ThermoFisher) and imaged in super-resolution using 3D structured illumination microscopy (3D-SIM) on a GE DeltaScan™ OMX SR microscope. Maximum intensity projections of volumetric image stacks are shown. Note in the side view of (A) that the turbidity of the tissue prevented imaging of all but the surface-most structures in the explant images, a common problem in imaging of both natural tissues and 3D cell cultures. Scale bars are 2 μm and apply to both top and side projections.

Article Snippet: CellWells were then immediately hydrated with PBS-1x solution, UV sterilized for 30 mins, and coated with 10 μg/ml each of purified human plasma fibronectin and human placenta collagen type VI (Rockland Immunochemical) for 30 minutes at 37 °C.

Techniques: Cell Culture, Staining, Microscopy, Imaging

Journal: STAR Protocols

Article Title: Protocol for Establishing Mouse Embryonic Stem Cells to Study Histone Inheritance Pattern at Single-Cell Resolution

doi: 10.1016/j.xpro.2020.100178

Figure Lengend Snippet:

Article Snippet: Fibronectin human plasma , Merck , Cat# F2006-1MG.

Techniques: Recombinant, Modification, Plasmid Preparation, Lysis, Protease Inhibitor, Transgenic Assay, Software, Imaging, Gel Extraction, Western Blot, Bicinchoninic Acid Protein Assay, Cell Culture, Luciferase, Reporter Gene Assay

Journal: STAR Protocols

Article Title: Protocol for Establishing Mouse Embryonic Stem Cells to Study Histone Inheritance Pattern at Single-Cell Resolution

doi: 10.1016/j.xpro.2020.100178

Figure Lengend Snippet:

Article Snippet: Fibronectin human plasma , Merck , Cat# F2006-1MG.

Techniques: Concentration Assay

Journal: The Journal of Immunology Author Choice

Article Title: Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

doi: 10.4049/jimmunol.1700128

Figure Lengend Snippet: Secretion of MMP-1, -10, and -7 by M. tuberculosis –infected primary monocytes is increased by adhesion to type I collagen and fibronectin. Monocytes in the presence or absence of type I collagen, or fibronectin were infected with M. tuberculosis (MOI = 1). For secretion analysis, supernatants were collected at 24 h poststimulation, whereas for gene expression cell lysates were collected at 6 h. MMP-1 concentrations were upregulated in M. tuberculosis –infected monocytes adherent to ( A ) type I collagen; ( B ) fibronectin, and MMP-1 mRNA accumulation was also upregulated in the presence of ( C ) type I collagen and ( D ) fibronectin. Samples were normalized to 18S rRNA. Secretion of ( E and F ) MMP-10, and ( G and H ) MMP-7 was also upregulated in the presence of type I collagen and fibronectin, after 24 h of M. tuberculosis infection. Graphs show means ± SD and are representative of three independent experiments performed in triplicate. ** p < 0.01, *** p < 0.001, **** p < 0.001. ns, not significant.

Article Snippet: Tissue culture plates were precoated with 100 μg/ml native type I collagen from human fibroblasts (VitroCol), 250 μg/ml type IV collagen from human placenta, or 20 μg/ml human plasma fibronectin (all from Advanced BioMatrix, Carlsbad, CA).

Techniques: Infection, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

doi: 10.4049/jimmunol.1700128

Figure Lengend Snippet: TIMP-1 and -2 secretion by M. tuberculosis –infected and CoMtb-stimulated monocytes adherent to type I collagen. Monocytes adherent to type I collagen, fibronectin or in the absence of matrix were either infected with M. tuberculosis (MOI = 1) or stimulated with CoMtb (1:5 dilution). Supernatants were collected at 24 h postinfection and analyzed for secreted concentrations of ( A ) TIMP-1 and ( B ) TIMP-2 with M. tuberculosis –infected monocytes, and ( C ) TIMP-1 and ( D ) TIMP-2 secretion of CoMtb-stimulated monocytes. CoMCont was used as control of CoMtb. Bars show mean ± SD and data are representative of three independent experiments performed in triplicate. *** p < 0.001, **** p < 0.001. ns, not significant.

Article Snippet: Tissue culture plates were precoated with 100 μg/ml native type I collagen from human fibroblasts (VitroCol), 250 μg/ml type IV collagen from human placenta, or 20 μg/ml human plasma fibronectin (all from Advanced BioMatrix, Carlsbad, CA).

Techniques: Infection

Journal: The Journal of Immunology Author Choice

Article Title: Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

doi: 10.4049/jimmunol.1700128

Figure Lengend Snippet: Surface expression of β3 and αV integrin subunits in M. tuberculosis –stimulated monocytes adherent to ECM proteins. Monocytes cultured in the presence or absence of type I collagen or fibronectin were stimulated for 24 h with CoMtb (1:5 dilution), and CoMCont as control, or directly infected with M. tuberculosis (MOI = 1). Cells were fixed, blocked, and stained with FITC-conjugated anti-integrin β1or primary anti-integrin β2, β3, or αV Abs and secondary FITC-conjugated anti-mouse IgG Ab. Secondary Ab alone or FITC-conjugated IgG1 isotype Ab were used as controls. ( A ) Histograms of integrin subunits β1, β2, and β3 for control and CoMtb-stimulated monocytes show an increase in β3 expression with CoMtb stimulation. MFIs of integrin subunits ( B ) β3 and ( C ) αV in monocytes stimulated with M. tuberculosis , and MFIs of ( D ) β3 and ( E ) αV in monocytes stimulated with CoMtb, in the presence or absence of matrix components ( n = 4). MFIs were normalized to baseline MFIs of respective controls (control media or CoMCont). (B)–(E) show data points and means. * p < 0.05.

Article Snippet: Tissue culture plates were precoated with 100 μg/ml native type I collagen from human fibroblasts (VitroCol), 250 μg/ml type IV collagen from human placenta, or 20 μg/ml human plasma fibronectin (all from Advanced BioMatrix, Carlsbad, CA).

Techniques: Expressing, Cell Culture, Infection, Staining