Journal: Virology

Article Title: The low pH-dependent entry of avian reovirus is accompanied by two specific cleavages of the major outer capsid protein mu 2C.

doi: 10.1006/viro.1996.0235

Figure Lengend Snippet: FIG. 8. Entry-related cleavages of m2C are cell type- and virus strain-independent. (A) Radiolabeled, purified avian reovirus strain 176 (176) and mammalian reovirus serotype 3 Dearing (T3) were used in an internalization assay, as described in the legend of Fig. 3, using QT6 cells or L929 cells, respectively. Samples were harvested at t Å 0, 45, or 90 min after virus adsorption and analyzed by SDS–PAGE and autoradiography. The locations of individual viral proteins are indicated on the sides of the figure. (B) Radiolabeled, purified avian reovirus strain S1133 was used in an internalization assay, as described in the legend of Fig. 3, using QT6 cells (QT6) or Vero cells (VERO). Samples were harvested at t Å 15, 30, 60, or 90 min after virus adsorption and analyzed by SDS–PAGE and autoradiography. The locations of individual viral proteins are indicated on the sides of the figure.

Article Snippet: The mammalian reovirus serotype 3 (Dearing strain)Since enveloped viruses utilize the same mechanism was obtained from Patrick Lee (University of Calgary)to mediate the membrane interactions involved in both and was plaque purified and grown in murine L929 cells.virus entry and syncytium formation (Bentz, 1993), avian Murine L929 cells (ATCC number 1-CCL) and Vero cellsreovirus entry was investigated to determine whether the (ATCC number 81-CCL) were obtained from the Ameri-neutral pH, fusion-inducing activity of s3 might bypass can Type Culture Collection.

Techniques: Virus, Purification, Adsorption, SDS Page, Autoradiography

Journal: Cell Proliferation

Article Title: RNAi‐mediated inhibition of the desmosomal cadherin (desmoglein 3) impairs epithelial cell proliferation

doi: 10.1111/j.1365-2184.2011.00765.x

Figure Lengend Snippet: Modulation of Dsg3 expression affected cell proliferation in MDCK cells. (a) Western blotting of cell lysates extracted from cells with either overexpression (knock‐in: KI) or knockdown (KD) of Dsg3. (b) Confocal microscopy of MDCK cells with ectopic Dsg3 expression [myc‐tag in (i)] or with Dsg3 knockdown (iii). Images in (ii) were vector control cells. (c) Dispase fragmentation assay of MDCK cells with either vector or Dsg3 shRNAi transduction. Cells were grown to confluence before being treated with 2.4 units/ml dispase for about 30 min to detach epithelial sheets. These sheets were subjected to mechanical stress by pipetting five times with 1 ml tips. Epithelial fragments were quantified by ImageJ and increased fragments (up to 2‐fold) were seen in cells with Dsg3 silencing by shRNAi‐1 or shRNAi‐2, respectively. Data are averages of duplicates in each group. (d) Growth curve of matched MDCK cells with up‐ or down‐regulation of Dsg3. Cells with overexpression had higher proliferation, but those with Dsg3 knockdown exhibited lower growth rate compared to matched control cells.

Article Snippet: HuSH TM shRNA plasmids (29‐mer) used for transduction in MDCK cells were purchased from OriGene Technologies Inc (Rockville, MD, USA).

Techniques: Expressing, Western Blot, Over Expression, Knock-In, Confocal Microscopy, Plasmid Preparation, Transduction

Journal: Chemical Science

Article Title: Semi-synthesis of a HGF/SF kringle one (K1) domain scaffold generates a potent in vivo MET receptor agonist † †Electronic supplementary information (ESI) available: Descriptions of reagents, antibodies, immunoblotting, MTT assay, scattering assay, morphogenesis assay and statistical analysis. See DOI: 10.1039/c4sc03856h Click here for additional data file.

doi: 10.1039/c4sc03856h

Figure Lengend Snippet: Cellular phenotypes induced by K1B/S. (A) Cell scattering assay. MDCK isolated cell islets were incubated for 18 h in culture media with 50 nM streptavidin (S), 50 nM anti-biotin antibody (Ab), 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. Cells were then stained and observed under microscope (40×). (B) Matrigel morphogenesis assay. MDCK cells were seeded onto a layer of Matrigel and treated for 18 h with 50 nM streptavidin (S), 50 nM anti-biotin antibody (Ab), 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. Cells were then observed under microscope (40×). (C) Angiogenesis. Mice were injected with a mixture of Matrigel and 1 nM HGF/SF (HGF), 10 nM VEGF, 100 nM NK1, 100 nM K1B/S, 100 nM K1B or 50 nM S. Hemoglobin absorbance was measured and concentration was determined using a rate hemoglobin standard curve and plug weight. ANOVA tests were performed to compare all the means, and a P -value < 0.001 was considered to indicate a statistically significant difference. (D) MTT assay. MDCK cells were cultured overnight (15 h) in medium containing 0.1% FBS with or without anisomycin (0.7 μM) and in the presence of 500 pM mature HGF/SF (HGF), 100 nM K1B, 100 nM K1B/S, 100 nM NK1 and 100 nM K1B/Ab. An MTT assay was then performed to evaluate cell survival. Results are expressed as the percentage of untreated control. An ANOVA test was performed to compare the 3 means, with a P -value < 0.05 considered statistically significant.

Article Snippet: Madin Darby Canine Kidney (MDCK, kind gift of Dr Jacqueline Jouanneau, Institut Curie, Paris, France) and Human cervical cancer HeLa cells, purchased from ATCC® (American Type Culture Collection, Rockville, MD, USA), were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Gibco, Karlsruhe, Germany), supplemented with 10% FBS (Fetal Bovine Serum, Gibco®, Life technologies, Grand Island, NY, USA) and 5 mL of ZellShieldTM (Minerva Biolabs GmbH, Germany).

Techniques: Scattering Assay, Isolation, Incubation, Staining, Microscopy, Injection, Concentration Assay, MTT Assay, Cell Culture, Control