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

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: Inhibitory effects of PA on inflammation induced by Aβ42 oligomer in mice. (A) TNF‐α, (B) IL‐1β, and (C) IL‐6 were examined using ELISA ( n = 6 per group). (D) Immunofluorescence analysis of Iba‐1, bar = 25 μm. (E) Western blots and (F) quantitative analysis of Iba‐1 ( n = 3 per group). All values are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control group. # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the AD model group.

Article Snippet: Blocking goat serum, Triton X‐100, DAPI nuclear staining, and antiquenching sealer for immunofluorescence analysis were purchased from Solarbio (Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Polygalacic acid attenuates cognitive impairment by regulating inflammation through PPARγ / NF‐κB signaling pathway

doi: 10.1111/cns.14581

Figure Lengend Snippet: NF‐κB expressions in the nucleus and cytoplasm after adding the inhibitor of PPARγ. (A) Western blots analysis for NF‐κB p65 subunit in nuclear and cytosolic and quantitative analysis of corresponding proteins. (B) The representative immunofluorescence images showing translocation of NF‐κB (p65, red) to the nucleus (blue) after treatment with Aβ42 oligomers or pretreat with PA or adding the inhibitor of GW9662. Bar = 50 μm. (C) Percent of nuclear NF‐κB p65‐positive cells. Values were expressed as the mean of at least three independent replicates, whose means resulted from 50 cells from three independent fields of view. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.001 compared with the Aβ42 stimulated group; & p < 0.05, && p < 0.01, &&& p < 0.001, and &&&& p < 0.001 compared with the PA treatment group. n = 3 for Western analysis, one‐way ANOVA with Tukey's post‐hoc test. Bars represent mean ± SD.

Article Snippet: Blocking goat serum, Triton X‐100, DAPI nuclear staining, and antiquenching sealer for immunofluorescence analysis were purchased from Solarbio (Beijing, China).

Techniques: Western Blot, Immunofluorescence, Translocation Assay, Control