Journal: PLOS Pathogens

Article Title: MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira , has an essential role during infection

doi: 10.1371/journal.ppat.1011313

Figure Lengend Snippet: (A) Predicted model of L . interrogans MPL36 with the double-psi beta-barrel domain (DPBB) (blue), a conserved region of RlpA proteins (green), and the SPOR domain (red) in the C-terminal. (B) Immunogold labeling of WT, Δ mpl36 , and Δ mpl36 + Leptospira strains were performed using polyclonal rabbit antiserum against MPL36 and goat anti-rabbit IgG labelled with 10 nm colloidal gold particles. Cells were visualized using 2% UA negative staining. (C) Whole intact spirochetes were incubated with different concentrations of Proteinase K (25–100 μg/mL), and western-blot analysis was conducted using polyclonal rabbit antisera against MPL36, LigA (positive control), and GroEL (negative control). (D) Recombinant proteins coated with fluorescent latex beads were incubated with immobilized MDCK cells and this interaction was assessed by fluorescent emission. LigA and LigB were used as positive control, while FlaA2 was used as negative control. The results are represented as mean ± standard deviation of two independent experiments.

Article Snippet: The cleavage fragments were analyzed by western blot with either mouse anti-human fibrinogen polyclonal antibody (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human vitronectin polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, rabbit anti-human laminin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human fibronectin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, or goat anti-human C3 polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, followed by peroxidase-conjugated anti-mouse (1:5,000), rabbit or goat (1:10,000) IgG (Sigma-Aldrich).

Techniques: Labeling, Negative Staining, Incubation, Western Blot, Positive Control, Negative Control, Recombinant, Standard Deviation

Journal: PLOS Pathogens

Article Title: MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira , has an essential role during infection

doi: 10.1371/journal.ppat.1011313

Figure Lengend Snippet: 96-well plates were coated with 1 μg of fibronectin (A), laminin (B), and PLG (C). 0.2 nmol of the recombinant proteins MPL36, LigA, LigB or FlaA2 coated with fluorescent latex beads were added per well, and the binding was assessed by fluorescent emission. A control using uncoated fluorescent latex beads was used to measure background signal. Data represent the mean ± standard deviation of results from three independent experiments (NS = non significant; *** p < 0.0001).

Article Snippet: The cleavage fragments were analyzed by western blot with either mouse anti-human fibrinogen polyclonal antibody (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human vitronectin polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, rabbit anti-human laminin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human fibronectin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, or goat anti-human C3 polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, followed by peroxidase-conjugated anti-mouse (1:5,000), rabbit or goat (1:10,000) IgG (Sigma-Aldrich).

Techniques: Recombinant, Binding Assay, Standard Deviation

Journal: PLOS Pathogens

Article Title: MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira , has an essential role during infection

doi: 10.1371/journal.ppat.1011313

Figure Lengend Snippet: (A) MPL36-bound PLG activation to plasmin (PLA) by urokinase-type PLG activator (uPA). 96-well plates were coated with 1 μg of rMPL36, rLigA, rLigB, rFlaA2 or BSA, and incubated with PLG (1 μg). After activation with uPA (4 ng) and the chromogenic substrate (S) (0.4 mM), the reaction was measured using a fluorometer. Data represent the mean absorbance value at 405 nm ± the standard deviation of three independent experiments (* p < 0.001). (B) Assessment of lysine residues involvement in MPL36/PLG interactions. PLG (10 μg/mL) was added to MPL36-coated wells (1 μg) in the presence of epsilon-aminocaproic acid (0.1–10 mM). Bound-PLG was detected with a polyclonal anti-PLG (1:2,000), followed by peroxidase-conjugated secondary antibodies (1:10,000). Data show the mean absorbance value at 492 nm ± the standard deviation of three independent experiments (* p < 0.05; ** p < 0.01). (C) Mapping of MPL36 region interacting with PLG by ligand affinity blot. Purified full-length (aa41-321) and truncated (aa41-305 and aa41-235) MPL36 recombinant proteins were subjected to SDS-12% PAGE under reducing conditions, transferred to a nitrocellulose membrane, and incubated with 50 μg of purified human PLG. Bound PLG was detected with anti-human PLG (1:500) followed by peroxidase-conjugated anti-rabbit IgG (1:10,000). BSA was included as a negative control.

Article Snippet: The cleavage fragments were analyzed by western blot with either mouse anti-human fibrinogen polyclonal antibody (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human vitronectin polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, rabbit anti-human laminin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, rabbit anti-human fibronectin polyclonal antibodies (Sigma-Aldrich) at a 1:5,000 dilution, or goat anti-human C3 polyclonal antibodies (Complement Technology) at a 1:5,000 dilution, followed by peroxidase-conjugated anti-mouse (1:5,000), rabbit or goat (1:10,000) IgG (Sigma-Aldrich).

Techniques: Activation Assay, Incubation, Standard Deviation, Purification, Recombinant, Negative Control

Journal: Bioengineering

Article Title: Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

doi: 10.3390/bioengineering7030089

Figure Lengend Snippet: Corneal fibroblasts organize fibronectin along compacted lines of fibrin fibers. HTK cells were cultured in a fluorescently labeled 3D fibrin matrix for 48 h in media containing PDGF BB (to stimulate cell spreading). Subsequently, cells were fixed and labeled for F-actin and fibronectin. Images are maximum intensity projections of z-series from each individual channel. Fibrin is compacted and aligned in front of the pseudopodial extensions, presumably due to tractional force generation. Fibronectin is organized into lines that colocalize with the compacted fibers (arrows). Scale bar is 20 µm.

Article Snippet: For immunostaining, a rabbit anti-human fibronectin polyclonal antibody (sc-9068) and a mouse anti-human fibronectin monoclonal antibody were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Cell Culture, Labeling

Journal: Bioengineering

Article Title: Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

doi: 10.3390/bioengineering7030089

Figure Lengend Snippet: Fibronectin tracks colocalize with compacted lines of fibrin fibers between cells. HTK cells ( A ) or NRK cells ( B ) were cultured in a fluorescently labeled 3D fibrin matrix for 48 h in media containing PDGF BB (to stimulate cell spreading). Subsequently, cells were fixed and labeled for F-actin and fibronectin. Images are maximum intensity projections of z-series from each individual channel. For both HTK and NRK cells, a network of fibronectin tracks is observed along and between groups of migrating cells. When present, fibronectin tracks colocalize with lines of fibrin compaction (arrows). In one area, fibrin matrix compaction is observed without corresponding fibronectin labeling (B, arrowhead). Scale bars are 150 µm.

Article Snippet: For immunostaining, a rabbit anti-human fibronectin polyclonal antibody (sc-9068) and a mouse anti-human fibronectin monoclonal antibody were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Cell Culture, Labeling

Journal: Bioengineering

Article Title: Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

doi: 10.3390/bioengineering7030089

Figure Lengend Snippet: Representative image of a living HTK cell showing fibronectin and fibrin organization in a 3-dimensional fibrin matrix. LifeAct-transfected cells were cultured overnight with media containing TGFβ1 to stimulate cell contractility. One hour before cells were placed in microscope for recording, fluorescent fibronectin was added to the media. Maximum intensity projections of z-series collected one hour after the start of the time-lapse are shown. Significant fibrin fiber compaction and alignment are present at both the front and rear of the cell (arrows). Compacted fibrin is coaligned with the intracellular F-actin stress fibers. Fibronectin labeling is limited to the cell area. Scale bar is 40 µm.

Article Snippet: For immunostaining, a rabbit anti-human fibronectin polyclonal antibody (sc-9068) and a mouse anti-human fibronectin monoclonal antibody were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Microscopy, Labeling

Journal: Bioengineering

Article Title: Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

doi: 10.3390/bioengineering7030089

Figure Lengend Snippet: Neighboring contractile cells create an interconnected network of compacted fibrin fibers, without the formation of a fibronectin network. HTK cells ( A ) or NRK cells ( B ) were cultured in a fluorescently labeled 3D fibrin matrix (cyan) for 48 h in media containing TGFβ1 (to stimulate cell contraction). Subsequently, cells were fixed and labeled for F-actin (red) and fibronectin (green). Images are maximum intensity projections of z-series from each individual channel. For both HTK and NRK cells, compacted lines of fibrin fibers developed between adjacent cells (arrows), forming an interconnected network. In contrast, fibronectin labeling was only observed where cells were present. Scale bars are 75 µm.

Article Snippet: For immunostaining, a rabbit anti-human fibronectin polyclonal antibody (sc-9068) and a mouse anti-human fibronectin monoclonal antibody were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Cell Culture, Labeling

Journal: Bioengineering

Article Title: Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

doi: 10.3390/bioengineering7030089

Figure Lengend Snippet: Correlation between actin, fibrin and fibronectin patterning. HTK cells ( A , B ) or NRK cells ( C , D ) were cultured in a fluorescently labeled 3D fibrin matrix for 48 h in media containing PDGF BB (to stimulate cell spreading) or TGFβ1 (to induce cell contractility). Subsequently, cells were fixed and labeled for F-actin and fibronectin. Correlation coefficients were calculated by comparing the relationship between actin, fibronectin, and fibrin fibers across the cell body and between neighboring cells. Across the cell body ( A , C ), high correlations were found for both PDGF BB and TGFβ1. Between neighboring cells ( B , D ), the only significant correlation found was between fibrin and fibronectin when cultured with PDGF BB (* three way ANOVA, p < 0.001). Means and standard deviations are calculated from three independent experiments (each with 5–10 neighboring cell pairs per culture condition).

Article Snippet: For immunostaining, a rabbit anti-human fibronectin polyclonal antibody (sc-9068) and a mouse anti-human fibronectin monoclonal antibody were used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Cell Culture, Labeling

Journal: Cells

Article Title: Fibronectin and Periostin as Prognostic Markers in Ovarian Cancer

doi: 10.3390/cells9010149

Figure Lengend Snippet: Immunohistochemical detection of fibronectin in ovarian cancer samples. ( A ) Images show representative examples of stromal fibronectin staining, scored as 1 (weak expression), 2 (moderate expression), and 3 (strong expression); ( B ) Images show a representative example of nuclear staining in cancer cells (image on the left) and lack of nuclear staining in cancer cells (image on the right); Pannoramic 250 Flash II Scanner, scale bar: 500 µm (upper panel) and 100 µm (lower panel).

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by normal horse blocking serum (2.5%; included in ImmPRESS Anti-Rabbit Ig Reagent Kit, MP-7401, Vector Laboratories, Burlingame, CA, USA) for 20 min. Then, sections were incubated with primary antibodies at 4 °C for 12 h. We used rabbit anti-human fibronectin polyclonal antibody (1:3000 dilution, A 0245, Dako, Glostrup, USA) and rabbit anti-human periostin polyclonal antibody (1:200 dilution, ab14041, Abcam, Cambridge, UK).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Fibronectin and Periostin as Prognostic Markers in Ovarian Cancer

doi: 10.3390/cells9010149

Figure Lengend Snippet: ( A ). Correlation between fibronectin expression and the anatomical source of tumor specimen (χ 2 test, p = 0.024) p —samples derived from peritoneal/omental metastatic disease; O—primary tumors (samples containing visible ovarian structures); T—samples containing only tumor tissue; ( B ). Examples of specimens derived from omental metastatic disease (P) with strong fibronectin expression; ( C ). Examples of specimens containing ovarian structures (O), green arrows indicate whitish corpuscles, red arrows indicate nests of cancer cells; ( D ). Correlation between fibronectin expression and the degree of desmoplastic reaction (χ 2 test, p < 0.001). *—denotes p < 0.05.

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by normal horse blocking serum (2.5%; included in ImmPRESS Anti-Rabbit Ig Reagent Kit, MP-7401, Vector Laboratories, Burlingame, CA, USA) for 20 min. Then, sections were incubated with primary antibodies at 4 °C for 12 h. We used rabbit anti-human fibronectin polyclonal antibody (1:3000 dilution, A 0245, Dako, Glostrup, USA) and rabbit anti-human periostin polyclonal antibody (1:200 dilution, ab14041, Abcam, Cambridge, UK).

Techniques: Expressing, Derivative Assay

Journal: Cells

Article Title: Fibronectin and Periostin as Prognostic Markers in Ovarian Cancer

doi: 10.3390/cells9010149

Figure Lengend Snippet: Kaplan-Meier analysis of overall survival and disease-free survival in 108 patients with advanced ovarian cancers stratified by stronger fibronectin expression (score 2 & 3) versus weak fibronectin expression (score 1) in the tumor stroma.

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by normal horse blocking serum (2.5%; included in ImmPRESS Anti-Rabbit Ig Reagent Kit, MP-7401, Vector Laboratories, Burlingame, CA, USA) for 20 min. Then, sections were incubated with primary antibodies at 4 °C for 12 h. We used rabbit anti-human fibronectin polyclonal antibody (1:3000 dilution, A 0245, Dako, Glostrup, USA) and rabbit anti-human periostin polyclonal antibody (1:200 dilution, ab14041, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cells

Article Title: Fibronectin and Periostin as Prognostic Markers in Ovarian Cancer

doi: 10.3390/cells9010149

Figure Lengend Snippet: Expression of fibronectin in tissue arrays. The y axis represents the number of samples. NAT—normal tissue adjacent to the tumor.

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by normal horse blocking serum (2.5%; included in ImmPRESS Anti-Rabbit Ig Reagent Kit, MP-7401, Vector Laboratories, Burlingame, CA, USA) for 20 min. Then, sections were incubated with primary antibodies at 4 °C for 12 h. We used rabbit anti-human fibronectin polyclonal antibody (1:3000 dilution, A 0245, Dako, Glostrup, USA) and rabbit anti-human periostin polyclonal antibody (1:200 dilution, ab14041, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cells

Article Title: Fibronectin and Periostin as Prognostic Markers in Ovarian Cancer

doi: 10.3390/cells9010149

Figure Lengend Snippet: Fibronectin and periostin analyzed altogether.

Article Snippet: Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by normal horse blocking serum (2.5%; included in ImmPRESS Anti-Rabbit Ig Reagent Kit, MP-7401, Vector Laboratories, Burlingame, CA, USA) for 20 min. Then, sections were incubated with primary antibodies at 4 °C for 12 h. We used rabbit anti-human fibronectin polyclonal antibody (1:3000 dilution, A 0245, Dako, Glostrup, USA) and rabbit anti-human periostin polyclonal antibody (1:200 dilution, ab14041, Abcam, Cambridge, UK).

Techniques: