Journal: BMC Cell Biology

Article Title: Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

doi: 10.1186/1471-2121-12-28

Figure Lengend Snippet: Role of the cytoskeleton in flotillin- 2 and PSGL-1 capping in human T-lymphoblasts in suspension . (A,B) After an overnight incubation in the absence of IL-2, T-lymphoblasts were incubated for 45 minutes at 37°C (control) or were preincubated for 30 minutes without or with latrunculin A (lat; 1 μM) or jasplakinolide (jasp; 5 μM) or blebbistatin (ble; 100 μM) or Y-27632 (Y; 10 μM) respectively followed by stimulation with 40 ng/ml of SDF-1 for 15 minutes at 37°C. Cells were then fixed with 10% TCA and (A) double-labeled either for flotillin-2 (flo2; rabbit Ab) and PSGL-1 (murine Ab) or (B) for flotillin-2 (flo2, rabbit Ab) and actin (murine Ab). Bar, 10 μm. (C-E) T-lymphoblasts were treated as described in (A) followed by evaluation of the % of polarized cells with contracted uropods (C); or evaluation of the % of cells with flotillin-2 caps (D) or evaluation of the % of cells with PSGL-1 caps (E). Mean ± s.e.m. of 3-7 independent experiments. At least 40 cells per sample and experiment were analyzed.

Article Snippet: Blebbistatin: Tocris Bioscience, UK.

Techniques: Suspension, Incubation, Control, Labeling

Journal: BMC Cell Biology

Article Title: Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

doi: 10.1186/1471-2121-12-28

Figure Lengend Snippet: Roles of myosin II and Rho-kinase in flotillin capping in human T-lymphoblasts adhering to fibronectin . A: After an overnight incubation in the absence of IL-2, T-lymphoblasts (1.5 × 10 6 cells per well) were placed on fibronectin-coated glass coverslips without or with blebbistatin (ble; 100 μM) or Y-27632 (Y; 10 μM) respectively and allowed to adhere for 30 minutes at 37°C. To part of the samples, 40 ng/ml of SDF-1 was added followed by a further 30 minutes incubation at 37°C (total incubation 60 minutes). Cells were then fixed with 10% TCA and double-labeled for flotillin-2 (flo2; rabbit Ab) and flo1 (murine Ab). Bar, 10 μm. Asterisks indicate cells shown enlarged in the panels labelled "detail". B, C: T-lymphoblasts were treated as described in (A) followed by evaluation of the % of polarized cells with contracted uropods (B); or evaluation of the % of cells with flotillin-2 caps (C). Mean ± s.e.m. of 3-4 independent experiments. At least 40 cells per sample and experiment were analyzed. * P < 0.025, ** P < 0.0125, *** P < 0.0005 for differences between data obtained for cells incubated in the absence and presence of inhibitors.

Article Snippet: Blebbistatin: Tocris Bioscience, UK.

Techniques: Incubation, Labeling

Journal: BMC Cell Biology

Article Title: Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

doi: 10.1186/1471-2121-12-28

Figure Lengend Snippet: Impact of pretreatment with blebbistatin on the lateral mobility of flotillin-2 cotransfected with flotillin-1 in freshly isolated T-lymphocytes

Article Snippet: Blebbistatin: Tocris Bioscience, UK.

Techniques: Isolation

Journal: The Journal of Cell Biology

Article Title: Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

doi: 10.1083/jcb.202007146

Figure Lengend Snippet: Imaging of junctional and apical defects with myo-IIA localization and inhibition by blebbistatin. (A) Deconvolution immunofluorescence staining of myo-IIA shows a similar organization to myo-IIB . Nonmuscle myo-IIA and myo-IIB are localized to aberrant apical contractile fibers. (B) Jeg-3 wild-type and knockout cells were treated with 25 µM blebbistatin for 30 min before immunofluorescence staining of ZO-1 and actin. Images are maximum-intensity projections of apical Z-slices as determined by ZO-1 signal. (C) Quantification of tight junction tortuosity using ZO-1 staining. Each point represents the tortuosity from one tight junction intersection to the next intersection. Center lines represent mean ± SEM; n ≥ 13. Scale bars, 5 µm in A, 10 µm in B.

Article Snippet: Calyculin A (catalog no. BML-El92-0100) was purchased from Enzo Life Sciences, and blebbistatin (catalog no. B592500) was purchased from Toronto Research Chemicals.

Techniques: Imaging, Inhibition, Immunofluorescence, Staining, Knock-Out

Journal: The Journal of Cell Biology

Article Title: Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

doi: 10.1083/jcb.202007146

Figure Lengend Snippet: pERMs negatively regulate myosin contractility. (A) Representative phase-contrast still images from showing the effect of 10 nM calyculin A on wild-type or knockout cells. (B) Same as A, except for 30-min treatment with 25 µM blebbistatin or transfection to express phosphomimetic ezrin-T567E before calyculin A treatment. Scale bars, 100 µm.

Article Snippet: Calyculin A (catalog no. BML-El92-0100) was purchased from Enzo Life Sciences, and blebbistatin (catalog no. B592500) was purchased from Toronto Research Chemicals.

Techniques: Knock-Out, Transfection

Journal: mBio

Article Title: Pseudomonas aeruginosa -Induced Bleb-Niche Formation in Epithelial Cells Is Independent of Actinomyosin Contraction and Enhanced by Loss of Cystic Fibrosis Transmembrane-Conductance Regulator Osmoregulatory Function

doi: 10.1128/mBio.02533-14

Figure Lengend Snippet: Role of actin contraction in P. aeruginosa -induced blebbing. (a) Representative images of blebbing (arrows) induced in human corneal epithelial cells following treatment with TNF-α and actinomycin D to induce apoptosis (upper panels) or infection with P. aeruginosa (MOI = 100) in gentamicin-containing media to kill extracellular bacteria (lower panels). Following 4 h of exposure to either the apoptotic cocktail or bacteria, 5 µM latrunculin A (or the DMSO control) was added for an additional 3 h prior to imaging. (b) Quantification of blebbing frequency in the experiment represented in panel a. Latrunculin inhibited apoptotic blebbing ( P = 0.001, Student’s t test) but not infection-induced blebbing ( P = 0.5, Student’s t test). NS, not significant. (c) Corneal epithelial cells were pretreated with either the r-enantiomer or the s-enantiomer of blebbistatin (40 µM) prior to inoculation with P. aeruginosa (MOI = 100), and blebbing frequency was quantified at 7 h postinoculation. ***, P < 0.001 (ANOVA with Dunnett’s multiple group comparison post hoc analysis comparing all groups to the untreated group). (d) Western blot analysis of phosphorylated myosin light chain 20 in corneal cells during the course of infection. (e) Rhodamine-phalloidin staining of corneal epithelial cells at various time points before and after infection with P. aeruginosa . All images were taken using the same exposure settings. A representative blebbing cell (the bleb had collapsed after fixation) shown in DIC (right) was free of stress fibers at 6 h postinfection.

Article Snippet: Blebbistatin enantiomers were purchased from Santa Cruz Biotechnology, Inc., reconstituted in DMSO to a stock of 40 mM, and used at a final concentration of 40 μM.

Techniques: Infection, Bacteria, Control, Imaging, Comparison, Western Blot, Staining