Journal: Experimental cell research

Article Title: Characterization of stitch adhesions: fibronectin-containing cell-cell contacts formed by fibroblasts

doi: 10.1016/j.yexcr.2019.111616

Figure Lengend Snippet: Immunofluorescence of CHX-treated fibroblasts incubated with full length FN or fragments of FN and stained for fibronectin (green) and actin (red). (A) The control CHX-treated cells did not stain with anti-fibronectin antibodies, indicating that synthesis and secretion of FN were completely blocked. (B) Prominent fibronectin stitches were observed between adjacent cells after incubation with full-length plasma FN. No organized FN was detected in samples incubated for the same period of time with 70kD (C) or 120kD (D) fibronectin fragments. Nuclei (blue) were stained with Hoechst. Insets present only fibronectin staining. Arrowheads in B indicate free cellular edges devoid of FN. Bar = 50μm.

Article Snippet: Statistics The average and standard deviation (SD) of immunofluorescence images and analysis by one way ANOVA were calculated using GraphPad Instat.

Techniques: Immunofluorescence, Incubation, Staining, Control, Clinical Proteomics

Journal: Experimental cell research

Article Title: Characterization of stitch adhesions: fibronectin-containing cell-cell contacts formed by fibroblasts

doi: 10.1016/j.yexcr.2019.111616

Figure Lengend Snippet: Dynamics of FN stitch development. (A) Pulse-chase analysis of the formation of stitch adhesions using labeled fibronectin preparations. CHX-treated fibroblasts were incubated with fibronectin labeled with green Alexa 488 (FN-488) for 1 hour, followed by another hour of incubation with fibronectin labeled with red Alexa 594 (FN-488/FN-594). Arrows indicate stitches formed during the second hour of incubation. Merged fluorescence and phase contrast images show localization of labeled stitches at places of cell-cell contacts. Nuclei were visualized with Hoechst (blue). Bar = 20μm. (B) Determination of stitch length. CHX-treated fibroblast were incubated with fibronectin for different periods, fixed and stained with anti-fibronectin antibodies. The lengths of FN stitches were measured on immunofluorescence images and presented as box plots.

Article Snippet: Statistics The average and standard deviation (SD) of immunofluorescence images and analysis by one way ANOVA were calculated using GraphPad Instat.

Techniques: Pulse Chase, Labeling, Incubation, Fluorescence, Staining, Immunofluorescence

Journal: Experimental cell research

Article Title: Characterization of stitch adhesions: fibronectin-containing cell-cell contacts formed by fibroblasts

doi: 10.1016/j.yexcr.2019.111616

Figure Lengend Snippet: Adhesion stitches aligned with actin filament bundles and associated with proteins typical of the integrin adhesome. Immunofluorescence images of (A) CHX-treated fibroblasts incubated with 25 μg/ml human plasma FN for 4 hours and then stained with anti-fibronectin antibodies (FN) and rhodamine-phalloidin (actin). Merged images (overlay) demonstrated alignment between actin bundles and fibronectin stitches (arrows). Fibronectin fibrils occasionally bridged the gap between neighboring cells (arrowheads). Bar = 20μm. Co-immunoprecipitation experiments of (B) CHX-treated cells in the absence (−FN) or presence (+FN) of fibronectin performed with anti-β1 integrin antibody 9EG7 (IP:9EG7) and rat IgG2a,κ isotype antibody control (IP:IgG), followed by Western blotting (WB) with antibodies against the indicated proteins revealed strong enrichment of adhesome proteins in β1 integrin complexes after incubation of cells with FN. Loading controls prior to immunoprecipitation (Lysate) are also shown.

Article Snippet: Statistics The average and standard deviation (SD) of immunofluorescence images and analysis by one way ANOVA were calculated using GraphPad Instat.

Techniques: Immunofluorescence, Incubation, Clinical Proteomics, Staining, Immunoprecipitation, Control, Western Blot