Journal: PLoS ONE

Article Title: A TNF Receptor 2 Selective Agonist Rescues Human Neurons from Oxidative Stress-Induced Cell Death

doi: 10.1371/journal.pone.0027621

Figure Lengend Snippet: ( A ) Differentiated LUHMES cells were incubated with different concentrations of hydrogen peroxide (H 2 O 2 ) for one hour. Then cells were washed with medium und cultivated for additional 24 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). ( B–E ) LUHMES cells were stimulated with H 2 O 2 (100 µM). After one hour the cells were washed with medium und regenerated for the indicated time intervals in medium with or without TNC-scTNF R2 (100 ng/ml). ( B ) LUHMES cells were regenerated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). ( C ) Cells were regenerated for one or three hours, fixed with 4%PFA, permeabilized with 0.1% Triton-X100 and β-III-tubulin was detected with specific antibodies. Cell nuclei were visualized using DAPI. Pictures are projections of eight optical sections (0.4 µm; bar = 50 µm). ( D ) Number of cells was determined by counting the nuclei (DAPI staining). ( E ) Cells were regenerated for one hour and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). (D,E) At least 10 different image sections containing a minimum of 500 cells were used to determine the number of total and TUNEL-positive cells. *p values less than 0.05 (** p-value less than 0.001) were considered to be significant (n = 2, shown are the mean values ± SD).

Article Snippet: The antibody anti-6-his was from Biovision (San Francisco, CA), the antibodies against huTNFR2 (HP9003; MR2-1) were from Hbt (Uden, The Netherlands), the antibody against huTNF (AF-210-NA) was from R&D Systems (Wiesbaden, Germany) and the antibody against β-III-tubulin was purchased from Acris Antibodies (Hiddenhausen, Germany).

Techniques: Incubation, MTT Assay, Staining, End Labeling, TUNEL Assay

Journal: PLoS ONE

Article Title: Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts

doi: 10.1371/journal.pone.0199412

Figure Lengend Snippet: The ability of the YH2CM to stimulate the Wnt or Notch signaling pathway was tested in cell lines by its ability to stabilize the β-catenin protein (for Wnt pathway) or Notch Intra-Cellular Domain protein (NICD, for Notch pathway). (A) L-cells were incubated with partially purified Wnt3a (0.2% and 0.5% v/v) or cYH2CM (10% and 50% v/v) for 4 hours before Western blotting for β-catenin and β-tubulin. The normalized β-catenin intensities were plotted indicating that cYH2CM does not stabilize β-catenin. (B) Western blot detecting β-catenin using mouse anti-human/mouse β-catenin monoclonal antibody (Clone 14/ β-catenin, BD Transduction Laboratories #610153) to probe the L-cell cell lysate. (C) HCT116 cells were incubated either with 50% (v/v) of DMEM (control) or cYH2CM for 1h, 3h and 72h. For EDTA treatment, the cells were incubated with EDTA/PBS (10 mM) for 15 min. Western blotting for NICD and β-tubulin was conducted on the cell lysate with the normalized NICD intensities plotted. cYH2CM does not stimulate NICD protein in β-catenin. (D) Western blot detecting NICD using rabbit anti-mouse cleaved Notch 1 monoclonal antibody (Clone D3B8, Cell Signaling #4147) to probe the HCT116 cell lysate. Error bars: SEM, n = 3, 2-way ANOVA Tukey’s test.

Article Snippet: The PVDF membranes were blocked in 5% (w/v) skim milk in TBS and the proteins of interest were detected using the following primary antibodies: mouse anti-human/mouse β-catenin monoclonal antibody (Clone 14/ β-catenin, BD Transduction Laboratories #610153), rabbit anti-mouse cleaved Notch 1 monoclonal antibody (Clone D3B8, Cell Signaling #4147) and the mouse anti-human/mouse β-tubulin monoclonal antibody (Clone 5H1, BD Pharmingen #556321); and secondary antibodies: IRDye® 800CW goat anti-rabbit polyclonal IgG (H+L) and IRDye® 800CW goat anti-mouse polyclonal IgG (H+L) (LI-COR Biosciences #926–32211 and 926–32210).

Techniques: Incubation, Purification, Western Blot, Transduction

Journal:

Article Title: Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

doi: 10.1152/ajplung.90577.2008

Figure Lengend Snippet: Stimulation of membrane adenylyl cyclase increases membrane-localized cAMP that does not disrupt microtubules in pulmonary microvascular endothelial cells (PMVECs). PMVECs were infected with a retrovirus to stably express GFP-α-tubulin. Live cell confocal imaging was performed for the first 5 min without treatment, and for another 30 min with forskolin (10 μmol/l) or isoproterenol (1 μmol/l). A: schematic representation of a retroviral construct that stably expresses GFP-α-tubulin. Microtubules did not reorganize in control- (B, n = 3), forskolin- (C, n = 5), or isoproterenol- (D, n = 5) treated cells. The scale bars represent 25 μm.

Article Snippet: Soluble proteins were suspended in 1× SDS-PAGE sample buffer (Invitrogen, Carlsbad, CA), resolved in 4–12% Bis-Tris gel (Invitrogen), transferred to nitrocellulose paper (Bio-Rad, Hercules, CA), and subjected to Western blot analysis using mouse monoclonal antibody to depolymerized β-tubulin (Covance, Berkeley, CA) or mouse monoclonal antibody to polymerized β-tubulin (Covance).

Techniques: Infection, Stable Transfection, Imaging, Construct

Journal:

Article Title: Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

doi: 10.1152/ajplung.90577.2008

Figure Lengend Snippet: Forskolin stimulation of sACI/II increases a cytosolic cAMP pool that reorganizes microtubules sufficient to disrupt PMVEC barrier function. PMVECs that are stably expressing GFP-α-tubulin were infected with an adenovirus expressing the sACI/II. Live cell confocal imaging was performed for 35 min in control cells and in forskolin (10 μmol/l) or isoproterenol (1 μmol/l) treated cells. A: in vehicle control cells, microtubules were not reorganized, and endothelial barrier was not disrupted (n = 4). B: in forskolin-treated cells, peripheral microtubule networks were reorganized, and endothelial cell gap formation (arrows denote endothelial barrier disruption as a consequence of reorganization of peripheral microtubule networks, n = 9). C: in isoproterenol-treated cells, microtubules were not reorganized, and the endothelial barrier was not disrupted (n = 5). The scale bars represent 25 μm.

Article Snippet: Soluble proteins were suspended in 1× SDS-PAGE sample buffer (Invitrogen, Carlsbad, CA), resolved in 4–12% Bis-Tris gel (Invitrogen), transferred to nitrocellulose paper (Bio-Rad, Hercules, CA), and subjected to Western blot analysis using mouse monoclonal antibody to depolymerized β-tubulin (Covance, Berkeley, CA) or mouse monoclonal antibody to polymerized β-tubulin (Covance).

Techniques: Stable Transfection, Expressing, Infection, Imaging

Journal:

Article Title: Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

doi: 10.1152/ajplung.90577.2008

Figure Lengend Snippet: Forskolin stimulation of sACI/II increases cytosolic cAMP that depolymerizes microtubules. PMVECs that are stably expressing GFP-α-tubulin were infected with an adenovirus expressing the sACI/II. Live cell confocal imaging was performed for 35 min in the absence (A) or presence (B) of forskolin (10 μmol/l). Western blot analysis was performed in these cells for depolymerized and polymerized tubulin, and live-cell tubulin labeling using Tubulin Tracker Green Reagent (250 nmol/l) was performed in vehicle-treated or forskolin (10 μmol/l)-treated PMVECs expressing sACI/II. A: in vehicle control cells, microtubules remained distributed throughout the cell, included at sites of cell-cell borders, over the 30-min time frame (n = 4). B: in forskolin-treated cells, fluorescence intensity of microtubules decreased significantly, in association with endothelial gap formation (n = 9). C: total fluorescence intensity decreased in control cells due to photobleaching over a 30-min time frame. Forskolin stimulation of sACI/II significantly decreased fluorescence intensity of microtubules, demonstrating microtubule reorganization and gap formation. *P < 0.05. D: forskolin stimulation of sACI/II increased depolymerized tubulin and decreased polymerized tubulin compared with control cells (n = 4). Summary is shown in E. *P < 0.05. F: forskolin stimulation of sACI/II decreased polymerized tubulin compared with control cells. The scale bars represent 25 μm. G: whole cell lysates were used to determine whether activation of sACI/II results in Tau-Ser214 phosphorylation. Whereas forskolin (10 μmol/l) increased Tau-Ser214 phosphorylation, isoproterenol (1 μmol/l) was without effect.

Article Snippet: Soluble proteins were suspended in 1× SDS-PAGE sample buffer (Invitrogen, Carlsbad, CA), resolved in 4–12% Bis-Tris gel (Invitrogen), transferred to nitrocellulose paper (Bio-Rad, Hercules, CA), and subjected to Western blot analysis using mouse monoclonal antibody to depolymerized β-tubulin (Covance, Berkeley, CA) or mouse monoclonal antibody to polymerized β-tubulin (Covance).

Techniques: Stable Transfection, Expressing, Infection, Imaging, Western Blot, Labeling, Fluorescence, Activation Assay

Journal:

Article Title: Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

doi: 10.1152/ajplung.90577.2008

Figure Lengend Snippet: Activation of sACI/II reversibly reorganizes microtubules necessary for intercellular gap formation. PMVECs were infected with an adenovirus expressing sACI/II, and the endogenous microtubule organization was examined using Tubulin Tracker Green Reagent (250 nmol/l). A: forskolin (10 μmol/l) stimulation of sACI/II reorganized endogenous microtubules and resulted in gap formation. Forskolin washout resulted in recovery of peripheral microtubule architecture over a 3-h time course. B: PMVECs were pretreated with paclitaxel (100 nmol/l) for 3 h, and then stimulated with forskolin (10 μmol/l) for 30 min. Paclitaxel stabilized microtubule structures and prevented forskolin from reorganizing microtubules and inducing intercellular gaps. The scale bars represent 25 μm. Arrows denote intercellular gaps.

Article Snippet: Soluble proteins were suspended in 1× SDS-PAGE sample buffer (Invitrogen, Carlsbad, CA), resolved in 4–12% Bis-Tris gel (Invitrogen), transferred to nitrocellulose paper (Bio-Rad, Hercules, CA), and subjected to Western blot analysis using mouse monoclonal antibody to depolymerized β-tubulin (Covance, Berkeley, CA) or mouse monoclonal antibody to polymerized β-tubulin (Covance).

Techniques: Activation Assay, Infection, Expressing