Journal: Advanced Healthcare Materials

Article Title: Curvature Perception of Mesenchymal Cells on Mesoscale Topographies

doi: 10.1002/adhm.202402865

Figure Lengend Snippet: Fluorescence images of pharmacologically manipulated hdF1 on different substrates. F‐actin in green, nuclei blue, and if stained microtubule in red. From left to right substrate curvature R is increasing, flat indicating substrate without topographies, the dashed lines point out topography boundaries. The first row shows hdF1 without (w/o) pharmacological manipulation, and row two hdF1 with impaired stress fiber formation by Blebbistatin treatment. The third row displays hdF1 with increased cell contractility due to RhoA/ROCK pathway activation by CN03. Row four shows hdF1 after microtubule depolymerization using Nocodazole, which also leads to RhoA activation. The last, fifth, row shows hdF1 with Taxol stabilized microtubule. Scale bars 100 µm.

Article Snippet: CN03 (Cytoskeleton, Inc.; dissolved in water;) was used to increase cell contraction via the Rho/ROCK pathway.

Techniques: Fluorescence, Staining, Activation Assay

Journal: Advanced Healthcare Materials

Article Title: Curvature Perception of Mesenchymal Cells on Mesoscale Topographies

doi: 10.1002/adhm.202402865

Figure Lengend Snippet: Effect of pharmacological manipulation on localization and orientation of isolated hdF1. A) Nucleus density (Mean ±SE). “Curv” indicates nucleus density on curved regions, “btw” nucleus density in transition zones, between the convex curved regions of the substrates (cf. Figure ). w/o displays hdF1 without treatment, other pharmacological manipulations as indicated. * Significant difference to w/o (p < 0.05). Notched box plots of B) Cell orientation, C) nucleus orientation, D) Cell perceived curvature, and E) Nucleus perceived curvature. Boxes show the 25th and 75th percentiles, the red lines indicate the median the black lines display the mean, and whiskers show the 5th and 95th percentile range. B–E only analyzed cells growing on the curved regions (curv). Range of cell/nucleus quantity assessed n w/o = /95‐118; n Blebbistatin = 98‐110; n CN03 = 115‐171; n Nocodazole = 98‐104; n Taxol = 100‐131; * Significant (p < 0.05), ** highly significant (p < 0.005), *** most significant (p < 0.001) to w/o or as indicated; ns not significant (p> 0.05) as indicated; Kruskal–Wallis with Dunn's posthoc test.

Article Snippet: CN03 (Cytoskeleton, Inc.; dissolved in water;) was used to increase cell contraction via the Rho/ROCK pathway.

Techniques: Isolation

Journal: Advanced Healthcare Materials

Article Title: Curvature Perception of Mesenchymal Cells on Mesoscale Topographies

doi: 10.1002/adhm.202402865

Figure Lengend Snippet: Pooled median perceived curvature of pharmacologically manipulated human dermal fibroblasts and corresponding nuclei. Pooled medians were calculated from all medians without significant difference as indicated in Figure 6D,E ; StDev stands for standard deviation. * Significant (p < 0.05), *** most significant (p < 0.001) difference to hdF without pharmacological manipulation (w/o), ( ** ) highly significant (p < 0.005); ( *** ) most significant (p < 0.001) difference between cell and nucleus perceived curvature, Kruskal‐Wallis with Dunn's posthoc test.

Article Snippet: CN03 (Cytoskeleton, Inc.; dissolved in water;) was used to increase cell contraction via the Rho/ROCK pathway.

Techniques: Standard Deviation, W/O

Journal: Advanced Healthcare Materials

Article Title: Curvature Perception of Mesenchymal Cells on Mesoscale Topographies

doi: 10.1002/adhm.202402865

Figure Lengend Snippet: Effect of curvature and pharmacological manipulation on cell spreading area and nucleus area. A) Schematic illustration of the microscopy projection bias correction. The scheme shows a cylinder in grey lines with a cell (green) spreading on it. The projection plane is visualized in light grey with dashed outlines. The visible projection by microscopy is indicated by the green element in the projection plane (dashed outline), whereas the orange element in the projection plane indicates the corrected cell outline. By manually specifying the radius of curvature R of the cylinder, the ImageJ plug‐in calculates the real distance b for every point of the cell outline (indicated by the blue x) with the measurable distance s to the cylinder axis. This procedure corrects the uniaxial distortion giving the new location of the cell outline point, visualized by the blue arrow heads in the projection plane. B) Notched Box plots of Cell spreading area (A CELL ) corrected for projection bias. C) Notched Box plots of Cell Aspect ratio (AR), corrected for projection bias. Aspect ratio = Major axis/Minor axis. D) Notched Box plots of Nuclei Area (A NUC ), corrected for projection bias. Boxes display 25 th and 75 th percentiles, the red lines the medians, and the black line the mean. B to D only analyzed cells growing on the curved regions (curv) of the substrates. * ( p < 0.05) significant, ** ( p < 0.005) highly significant, ***(p < 0.001) most significant to w/o; * significant (p < 0.05), ** (p < 0.00 5) highly significant, *** (p < 0.001) most significant as indicated; ns not significant (p> 0.05) as indicated; Kruskal–Wallis with Dunn's post‐hoc test. Range of cell/nucleus quantity assessed n w/o = 84–118; n Blebbistatin = 33‐99; n CN03 = 114‐171; n Nocodazole = 77‐104; n Taxol = 100‐131; Sample size aspect ratio: n w/o = 49‐69; n Blebbistatin = 34‐82; n CN03 = 45‐88; n Nocodazole = 45‐68; n Taxol = 54‐81.

Article Snippet: CN03 (Cytoskeleton, Inc.; dissolved in water;) was used to increase cell contraction via the Rho/ROCK pathway.

Techniques: Microscopy

Journal: The Journal of Cell Biology

Article Title: Cyclin A2 modulates kinetochore–microtubule attachment in meiosis II

doi: 10.1083/jcb.201607111

Figure Lengend Snippet: MT dynamics are altered in cyclin A2 knockout oocytes. (a) Representative time-lapse images of MII-stage cyclin A2 fl/fl and cyclin A2 −/− oocytes. PAGFP-α-tubulin (green) was photoactivated (PA) using a 405-nm laser, and the time course of PAGFP fluorescence decay was monitored. Spindle MTs were visualized using X-rhodamine (red), and chromosomes were visualized using Hoechst 33342 (blue). Bars, 2 μm. (b) Normalized fluorescence decay curves obtained by measuring the loss of PAGFP fluorescence in cyclin A2 fl/fl ( n = 7) and cyclin A2 −/− oocytes ( n = 8). Decay plots show fast and slow components assumed to represent non-kMTs and kMTs, respectively. Fluorescence decay after Taxol treatment is used to determine the bleaching coefficient for each time point. Error bars represent SEM. (c) Calculated half-life of fast (non-kMTs) and slow (kMTs) shows that MTs have a longer half-life in cyclin A2 −/− oocytes. Error bars represent SEM. *, P < 0.05 by t test.

Article Snippet: X-rhodamine–labeled tubulin protein (Cytoskeleton, Inc.) was dissolved in general tubulin buffer (Cytoskeleton, Inc.) containing 1 mM GTP sodium salt (Cytoskeleton, Inc.).

Techniques: Knock-Out, Fluorescence

Journal:

Article Title: Novel Suicide Ligands of Tubulin Arrest Cancer Cells in S-Phase 1

doi:

Figure Lengend Snippet: Microtubule polymerization inhibition by IAABE and BAABE. Compounds were dissolved in G-PEM buffer at 4°C before resuspending the lyophilized tubulin in the wells of a 96-well plate (CytoDYNAMIX™ Screen 01; Cytoskeleton Inc.). Absorbance was measured over time; absorbance is proportional to microtubule content. IC50 values were determined by linear regression analysis of concentration versus percentage inhibition; concentration at 50% inhibition at 60 minutes was defined as the IC50. Concentrations of IAABE (A) were 0 (■), 1.0 (♦), 2.5 (▲), 5.0 (□) and 10 µM (○). Concentrations of BAABE (B) were 0 (■), 10 (♦), 20 (▲), 40 (□) and 80 µM (○).

Article Snippet: Compounds were dissolved in G-PEM buffer at 4°C before resuspending the lyophilized tubulin in the wells of a 96-well plate (CytoDYNAMIX ™ Screen 01; Cytoskeleton Inc.).

Techniques: Inhibition, Concentration Assay

Journal: Microorganisms

Article Title: Equine Alphaherpesviruses Require Activation of the Small GTPases Rac1 and Cdc42 for Intracellular Transport

doi: 10.3390/microorganisms8071013

Figure Lengend Snippet: Small GTPase activation with ratiometric FRET in response to EHV-1 infection. Raichu-FRET biosensors were used to examine the activation status of small GTPases. ED cells were transfected with pRaichu-Rac1- or pRaichu-Cdc42-biosensor expressing plasmids. Twenty-four hours after transfection, EHV-1 RFP was added to ED cells and emissions of donor and acceptor was simultaneously measured and later analyzed. The ratio of FRET/CFP (acceptor/donor) was calculated for the selected region of interest (transfected cell) and plotted as a moving average of 20 frames.

Article Snippet: RhoA Inhibitor I based on a purified C3 Transferase (dissolved in water; Cat. # CT04, Cytoskeleton, Inc.), and a Rho/Rac/Cdc42 Activator I (dissolved in water; Cat. # CN04, Cytoskeleton, Inc.) were used at final concentrations of 2 μg/mL, following the manufacturer’s instructions.

Techniques: Activation Assay, Infection, Transfection, Expressing

Journal: Microorganisms

Article Title: Equine Alphaherpesviruses Require Activation of the Small GTPases Rac1 and Cdc42 for Intracellular Transport

doi: 10.3390/microorganisms8071013

Figure Lengend Snippet: Effects of small GTPase inhibitors on virus transport. Inhibitor-treated ED cells were infected with EHV-1 at a MOI of 1, fixed at 30 min, 3 or 6 h after infection, and stained in one of the following ways. ( A ) ED cells fixed at 30 min post infection were stained with FITC-lectin, and the number of particles localizing with the plasma membrane was counted manually. ( B ) At 6 hpi, ED cells were fixed and stained with DAPI. ( C – E ) Colocalization of EHV-1 with endosomal markers in ED cells treated with inhibitors of the small GTPases Cdc42 and Rac1. Colocalization of EHV-1 with cellular compartments: cells were stained with only DAPI at 6 hpi. Exemplary images following staining: ( F ) DAPI stain at 6 hpi. ( G ) FITC-lectin 30 min after infection, ( H ) anti-LAMP-1 antibodies 3 hpi, ( I ) anti-EEA-1 antibodies 3 hpi, ( J ) or anti-caveolin antibodies 3 hpi. Scale bars represent 10 μm. DAPI is pseudocolored blue, EHV-1 particles as red, staining target (Lectin, LAMP-1, EEA-1, or Caveolin-1) as green. Data are presented as means with S.D. of three independent experiments and normalized to the mean (with S.D.) of the control (virus infection without inhibitor treatment). One-way ANOVA was employed, followed by Dunnett’s multiple comparisons correction; * p < 0.05.

Article Snippet: RhoA Inhibitor I based on a purified C3 Transferase (dissolved in water; Cat. # CT04, Cytoskeleton, Inc.), and a Rho/Rac/Cdc42 Activator I (dissolved in water; Cat. # CN04, Cytoskeleton, Inc.) were used at final concentrations of 2 μg/mL, following the manufacturer’s instructions.

Techniques: Infection, Staining

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: Environmental scanning electron microscope image of adsorbed rhodamine-labeled laminin cluster on pristine TNSs ( left ). Proteins were subsequently stained orange. Adsorbed amount of laminin determined after 30 min for four different initially applied protein concentrations ( right ). On all TNSs, increasing the protein mass in solution leads to a higher amount of adsorbed proteins. Elevating C fluence results in impediment to protein adsorption.

Article Snippet: For dissolved rhodamine-labeled laminin (LMN01, Cytoskeleton Inc., Denver, CO, USA) under neutral conditions, the ζ -potential and hydrodynamic radius of the protein was determined with dynamic light scattering experiments using a Malvern Zetasizer (Zetasizer Nano ZS, Malvern Panalytical GmbH, Kassel, Germany).

Techniques: Microscopy, Labeling, Staining, Adsorption

Journal: Nanomaterials

Article Title: Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

doi: 10.3390/nano12213858

Figure Lengend Snippet: ζ -potential and mean relative laminin adsorption for examined TNSs. Protein adsorption experiments on TNSs were performed for four laminin concentrations (0.1, 0.5, 1.0, and 1.5 μ g·cm − 2 ). Ion fluences are given in ions·cm − 2 .

Article Snippet: For dissolved rhodamine-labeled laminin (LMN01, Cytoskeleton Inc., Denver, CO, USA) under neutral conditions, the ζ -potential and hydrodynamic radius of the protein was determined with dynamic light scattering experiments using a Malvern Zetasizer (Zetasizer Nano ZS, Malvern Panalytical GmbH, Kassel, Germany).

Techniques: Adsorption