Journal: Frontiers in Cell and Developmental Biology

Article Title: An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes

doi: 10.3389/fcell.2021.712627

Figure Lengend Snippet: Indirect Immunofluorescence of focal adhesions in DITNC1 cells. (A) Image to the top shows the tissue culture plate with the magnets, which create the magnetic field. The scheme illustrates details of the experimental approach. Cells were plated on coverslips (light blue surface) and incubated with serum-free medium for 30 min. DITNC1 cells (beige) were stimulated with TRAIL-R2- (as a negative control) or Thy-1-coated Protein A-conjugated magnetic beads (blue spheres with yellow sticks), with or without mechanical stress (MS) applied by a magnet (gray) for 5 min. (B) Focal Adhesions (FA) were stained with an antibody against phospho-tyrosine, followed by a secondary antibody. Magnification bar = 10 μm. (C,D) Values in the graph are the mean + s.e.m. from at least 30 cells that were measured per condition in each experiment ( n = 3) of the number (N°) of FA per cell (C) and the average area (μm 2 ) of the FA (D) . Statistical significance was calculated using the Kruskal–Wallis non-parametric test with Dunn’s multiple comparison. # p < 0.05, compared with the Thy-1 condition.

Article Snippet: The rat astrocytic cell line DITNC1 (ATCC CRL-2005) was obtained from P. Magistretti (University of Lausanne, Switzerland), maintained in RPMI medium 1640 (GIBCO, Pittsburgh, PA, United States) containing 5% fetal bovine serum (FBS, HyClone, Pittsburgh, PA, United States), 0.1 mM 2-mercaptoethanol (GIBCO) and 100 U/ml penicillin/100 μg/ml streptomycin (PS mixture, GIBCO).

Techniques: Immunofluorescence, Incubation, Negative Control, Magnetic Beads, Staining, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes

doi: 10.3389/fcell.2021.712627

Figure Lengend Snippet: Indirect Immunofluorescence of stress fibers in DITNC1 cells. (A) Cells were plated on coverslips and incubated with serum-free medium for 30 min, and then stimulated as indicated in , for 5 min. Stress fibers were labeled with phalloidin/FITC (green). Magnification bar = 8 μm. Values in the graph are (B) Number of stress fibers per cell, (C) Average thickness (μm) of stress fibers per cell, and (D) Stress fiber coherency (alignment) per cell. Values in histograms and error bars represent mean + s.e.m. from at least 30 cells that were measured per condition in each experiment ( n = 3). Statistical significance was calculated using Kruskal–Wallis non-parametric test with Dunn’s multiple comparison. MS, Mechanical Stress. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with basal conditions.

Article Snippet: The rat astrocytic cell line DITNC1 (ATCC CRL-2005) was obtained from P. Magistretti (University of Lausanne, Switzerland), maintained in RPMI medium 1640 (GIBCO, Pittsburgh, PA, United States) containing 5% fetal bovine serum (FBS, HyClone, Pittsburgh, PA, United States), 0.1 mM 2-mercaptoethanol (GIBCO) and 100 U/ml penicillin/100 μg/ml streptomycin (PS mixture, GIBCO).

Techniques: Immunofluorescence, Incubation, Labeling, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes

doi: 10.3389/fcell.2021.712627

Figure Lengend Snippet: Phosphorylation of Myosin Light Chain after Thy-1 stimulation, with or without mechanical stress. DITNC1 cells were incubated with serum-free medium for 30 min and stimulated as indicated in for 5, 10, 15, and 20 min. (A,C) Whole cell lysates were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Bands of 18 kDa correspond to Myosin Light Chain phospho-Serine 19 (p-MLC) and those at 90 kDa, to the Heat Shock Protein 90 (HSP90). Blue and red scissors indicate where the figure (immunoblot image) was cut so that bands match the order of sample appearance in the corresponding graphs. (B,D) Values in the graphs correspond to densitometric analysis of the p-MLC band intensity normalized to Heat Shock Protein 90 (HSP90) values. The application of mechanical forces exerted by a magnet are indicated using a gray shaded background in graph D. Statistical significance was calculated using Mann-Whitney non-parametric test. * and #, p < 0,05; *compared with the basal condition at T = 0, # compared with the value obtained at the same time with TRAIL-R2.

Article Snippet: The rat astrocytic cell line DITNC1 (ATCC CRL-2005) was obtained from P. Magistretti (University of Lausanne, Switzerland), maintained in RPMI medium 1640 (GIBCO, Pittsburgh, PA, United States) containing 5% fetal bovine serum (FBS, HyClone, Pittsburgh, PA, United States), 0.1 mM 2-mercaptoethanol (GIBCO) and 100 U/ml penicillin/100 μg/ml streptomycin (PS mixture, GIBCO).

Techniques: Phospho-proteomics, Incubation, SDS Page, Western Blot, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes

doi: 10.3389/fcell.2021.712627

Figure Lengend Snippet: Traction forces are increased after Thy-1 and mechanical stress stimulation in astrocytes. (A) Representative images of DITNC1 cells stimulated only with Protein A-magnetic beads coated with Thy-1 (top row) or with Thy-1-Protein A-magnetic beads plus mechanical stress (MS) (bottom row). The left column corresponds to Reflection Interference Contrast Microscopy (RICM), the middle column shows Bright Field (BF), and the right column, Total Reflection Internal Fluorescence of Traction Forces (TRITC). Magnification bar = 12 μm (B) Quantification of mean fluorescence intensity measuring traction force for the basal, Thy-1 alone (for 5 min) and Thy-1 + mechanical stress (for 5 min) conditions (at least 30 cells per condition were counted). Values were normalized to the fluorescence intensity observed for cells without bead contact at the same time point for each condition (value + s.e.m. of three independent experiments). Statistical significance was calculated using the Kruskal–Wallis non-parametric test, with Dunn’s multiple comparison. **** p < 0.0001, compared with basal tension; #### p < 0.0001, compared with the tension corresponding to Thy-1 only.

Article Snippet: The rat astrocytic cell line DITNC1 (ATCC CRL-2005) was obtained from P. Magistretti (University of Lausanne, Switzerland), maintained in RPMI medium 1640 (GIBCO, Pittsburgh, PA, United States) containing 5% fetal bovine serum (FBS, HyClone, Pittsburgh, PA, United States), 0.1 mM 2-mercaptoethanol (GIBCO) and 100 U/ml penicillin/100 μg/ml streptomycin (PS mixture, GIBCO).

Techniques: Magnetic Beads, Microscopy, Fluorescence, Comparison