Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. E1A expression at 2 hr pi as measured after virus infection alone (V), or infection following bafilomycin A pretreatment (Baf+V) at 250 nM or 500 nM, and in the solvent control (DMSO+V). B. Effect on E1A mRNA expression of pretreatment with cytochalasin D prior to virus infection (Cyto D+V) at 250 nM or 2 μM; solvent control (DMSO+V), virus alone (V); 2 hr pi (DMSO+V vs. Cyto D+V 2 μM; *p<0.05, ANOVA). C. Effect on E1A mRNA expression of pretreatment with nocodazole prior to viral infection (Noc+V) at 10 μM or 30 μM, virus alone (V), and DMSO control (DMSO+V). D. Confocal microscopy representation of viral entry at 2 hr pi in the presence of solvent control (panel 1, DMSO+V) or 30 μM nocodazole (panel 2, Noc+V). Cy3 labelled HAdV-D37 (red), anti-α tubulin staining (green). E. Western blot showing protein expression levels in keratocytes after dynamin 2 (DNM2) knockdown: mock transfected and virus infected (V), scRNA transfected and virus infected (scRNA+V), siDNM2 transfection prior to viral infection (siDNM2+V). Western blot for α-AP2Al, an adaptor protein shown as negative control for specificity of DNM2 knockdown. Actin blot shows load control. Dynamin 2 mRNA expression levels by qRT-PCR are represented in bar graph just below. F. Early gene expression (E1A) as measured by qRT-PCR from same RNA pool as in E: virus alone (V), scrambled RNA (scRNA+V), dynamin 2 knockdown, virus infected cells (siDNM2+V) (*p<0.001, ANOVA). G. Western blot shows expression of mCherry in empty vector prior to virus infection (EV+V) and mCherry-dynamin 2 (arrow) prior to virus infection (OE-DNM2+V). The bar graph just below shows mRNA expression of dynamin 2. H. E1A mRNA expression as measured in cells after dynamin 2 vector pretreatment, performed on same RNA pool from G (*p=.01, Students t test). Error bars represent standard deviation of the mean. Each experiment was repeated at least 5 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Expressing, Virus, Infection, Solvent, Control, Confocal Microscopy, Staining, Western Blot, Knockdown, Transfection, Negative Control, Quantitative RT-PCR, Gene Expression, Plasmid Preparation, Standard Deviation

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Western blot for dynamin 2, acetylated tubulin, and GAPDH after mock infection (M), virus infection (V), empty vector transfection prior to virus infection (EV+V), and dynamin 2 vector transfection prior to infection (OE-DNM2+V). Blots were cut at the appropriate sizes and probed with specific antibodies. B. Western blot for dynamin 2 protein, acetylated tubulin, and GAPDH in cells after treatment with scRNA or dynamin 2 siRNA prior to viral infection (scRNA+V, siRNA+V, respectively). C. Confocal microscopy of mock and Cy3-labeled (red) virus infected cells, 1 hr pi, stained for acetylated tubulin (green). Mock infected and virus infected cells both show increased perninuclear acetylated tubulin when knocked down for dynamin 2 (row 2, right hand column, arrows, and row 4, right hand column). Virions in cells knocked down for dynamin 2 appear to accumulate in the perinuclear region at 1 hr pi. Each experiment was repeated at least 5 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Western Blot, Infection, Virus, Plasmid Preparation, Transfection, Confocal Microscopy, Labeling, Staining

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Modified schematic of SLO assay (Suomalainen et al., 2013). In this assay, SLO permeabilizes only cell membranes and not endosomal membranes, so that Cy3-labeled virions within endosomes appear red, while virions in the cytosol appear yellow upon binding to anti-Cy3 antibody with a green chromophore. B. Streptolysin O (SLO)-penetration assay performed 1 hr pi. Panel 1 shows the no-SLO control, where there is no staining for GM-130, indicating absence of antibody penetration. Panels 2-5 show equivalent staining for GM130 (green), indicating that the transfections did not affect pore formation by SLO. C. SLO assay performed in cells transfected prior to viral infection with empty vector (EV), dynamin 2 overexpression construct (OE-DNM2), scRNA, or siRNA (siDNM2). In SLO treated groups (+SLO), virions in endosomes appear red, while virions in cytosol appear yellow. As a control, left panel shows same groups treated with triton X100 without SLO (−SLO), and thus virions in both cytosol and endosomes appear yellow. Each experiment was repeated at least 3 times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Modification, Labeling, Binding Assay, Control, Staining, Transfection, Infection, Plasmid Preparation, Over Expression, Construct

Journal: Virology

Article Title: Impact of dynamin 2 on adenovirus nuclear entry

doi: 10.1016/j.virol.2019.01.008

Figure Lengend Snippet: A. Confocal microscopy of cells pretreated with empty vector (EV), dynamin 2 expressing vector (OE-DNM2), scRNA, or dynamin 2 siRNA (siDNM2) and infected with Cy34abeled HAdV-D37 (red) for 1 hr, and stained with anti-pericentrin (green), and DAPI (blue). B. Distance from green signals (arrows) to the closest edge of adjacent nuclei (100 cells per experimental group in three experiments) was measured using Leica application suite X (LAS X, Leica Microsystems, Wetzlar, Germany), and graphed in boxplots (*p<0.0001, Kruskal-Wallis). C. Transmission electron microscopy of infected keratocytes from same pretreatment groups, with 3 distinct images shown per group shown. NU: nucleus. Scale bar = 500 nm. D. Measurements taken from each microtubule organizing center to nearest nuclear membrane were performed on 10 cells per group by a masked observer. Data shown reflects means and standard deviation for each group (*p<0.05, Students t test). E. Confocal microscopy showing co-localization (yellow) of nuclear pore complex protein Nup358 (green), and Cy3-labeled HAdV-D37 (red), 2 hr pi. Each experiment was performed three times with similar results.

Article Snippet: Cy3 dye was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, cat. no. PA23031).

Techniques: Confocal Microscopy, Plasmid Preparation, Expressing, Infection, Staining, Transmission Assay, Electron Microscopy, Membrane, Standard Deviation, Labeling

Journal: Oncogene

Article Title: α3β1 integrins regulate CD151 complex assembly and membrane dynamics in carcinoma cells within 3D environments.

doi: 10.1038/onc.2012.415

Figure Lengend Snippet: Figure 6. a3 integrin promotes protrusion in cells in 3D ECM. (a) Representative images of cells expressing control or a3 integrin shRNA were co-transfected with Lifeact–GFP (LA–GFP) and CD151–RFP and embedded in 3D native collagen I (COL I) or matrigel (MG) within imaging chambers. Cells were left to invade for 24 h and were then fixed and subjected to confocal microscopy. (b) Representative confocal z-stack projections of control cells expressing Lifeact–GFP and embedded within cy3-labelled COL or MG 3D gels. (c) Representative 3D projections of confocal z-stacks of control or a3 integrin shRNA cells expressing Lifeact–GFP and invading into 3D COL or MG gels. Images are stills taken from movies shown in Supplementary data. (d) Analysis of dynamics of Lifeact–GFP protrusions from cells as shown in example images in c. Protrusion number per cell was calculated for each time point over the duration of the movie (d). n ¼ 8 cells per condition over three independent experiments. (e) Graph showing change in protrusion volume as a % of total cell volume over time from analysis of movies as in (d). (f) Protrusion: retraction ratio as calculated from protrusion dynamics data from live cell analysis as in d and e. *P ¼ 0.01, **P ¼ 0.005 compared with shCon. Scale bars, 10 mm.

Article Snippet: 1 ml of diluted collagen or laminin was added to an aliquot of Cy3 label from a Cy3 Mono-Reactive Dye Pack (GE Healthcare, Bucks, UK) and mix/rotated for 30 min at 41C in the dark.

Techniques: Expressing, Control, shRNA, Transfection, Imaging, Confocal Microscopy, Cell Analysis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mechanisms of the lysophosphatidic acid-induced increase in [Ca 2+ ] i in skeletal muscle cells

doi: 10.1111/j.1582-4934.2008.00139.x

Figure Lengend Snippet: Effect of lysophosphatidic acid receptor antagonists and G i protein inhibitor on LPA-induced changes in intracellular Ca 2+ concentration in C2C12 cells. C2C12 cells were pre-treated with 10 μM of the LPA receptor antagonists, VPC 12249, VPC 32183 and DGPP (8.0), for 10 sec or with pertussis toxin (PTX, 100 ng/ml), a G i protein inhibitor, for 10 min prior to the addition of LPA (10 μM). The [Ca 2+ ] i was measured as described in the Materials and methods. Values are means ± S.E.M. of six different experiments. *P < 0.05 versus vehicle control value. DGPP: dioctanoyl glycerol pyrophosphate; LPA : lysophosphatidic acid, [Ca 2+ ] i : intracellular calcium concentration.

Article Snippet: In order to determine if the LPA-induced increase in [Ca 2+ ] i was a LPA-receptor mediated response, C2C12 cells were pre-treated with LPA 1/3 receptor antagonists, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 10 μM), VPC12249 (10 μM) or VPC 32183 (10 μM) [ ] (Avanti Polar Lipids, Inc, Al, USA) for 10 sec before the addition of LPA.

Techniques: Concentration Assay, Control