Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Analysis of C15 infectivity by immunofluorescent staining. HBEC-ALI cultures were infected for 18 h with C15 and imaged apically and z-stacked orthogonally by confocal microscopy. Secretory and ciliated cells were stained with WGA and rabbit polyclonal antibody against aat, respectively ( false color red ), C15 capsid stained with monoclonal antibody ( false color green ), nuclei stained with Syto-13 ( false color blue ). Scale bars indicate 20 μm

Article Snippet: Preliminary experiments revealed that cells cultured using a different HBEC culture medium (PneumaCultTM, Stem Cell Technologies, Vancouver, Canada) had more plentiful ciliated cells [ ].

Techniques: Infection, Staining, Confocal Microscopy

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Single cell analysis of RV-C infection and epithelial cell surface markers. HBEC-ALI cultures infected or mock-infected for 18 h with C15 or BEGM alone, respectively, were labeled with antibodies against C15 capsid (C15), and either a acetylated-alpha-tubulin (aat, Cilia) or b fluorescently labeled-WGA (WGA) and analyzed by flow cytometry. The graphs summarize the percentage of C15+ cells in ciliated and non-ciliated cells ( n = 4 independent experiments, two cell donors). Mock, mock-inoculated cultures; C15, C15-inoculated cultures. *** p < 0.001

Article Snippet: Preliminary experiments revealed that cells cultured using a different HBEC culture medium (PneumaCultTM, Stem Cell Technologies, Vancouver, Canada) had more plentiful ciliated cells [ ].

Techniques: Single-cell Analysis, Infection, Labeling, Flow Cytometry

Journal: Life Science Alliance

Article Title: RAS and PP2A activities converge on epigenetic gene regulation

doi: 10.26508/lsa.202301928

Figure Lengend Snippet: (A) Cell viability inhibitory effect of increasing concentrations of panobinostat (HDACi) in PP2A-A–depleted cells as compared to siCtrl (n = 3). (B, C) Synergy between panobinostat and the PP2A activator DBK1154 as a dose–response matrix and with Bliss synergy area, respectively (n = 2). (D) Western blots showing the total protein levels of HDAC1/2 in whole-cell lysates of cells after RAS or PP2A manipulation. Left panel: siRNA-mediated inhibition of PP2A inhibitory proteins and RAS for 72 h in H460 cells; middle panel: pharmacological PP2A activation by drug treatment (10 µM, 48 h); and right panel: HBEC step-wise–transformed by shTP53 (p53−), mutant KRAS expression (KRAS+), and small-t–mediated PP2A inhibition (ST+). (E) Validation of the suitability of the reporter system for detecting gene activation upon pharmacological targeting of PP2A/RAS-regulated epigenetic mechanisms. Shown are relative amounts of GFP-positive HCT116 SFRP1-GFP reporter cells by IncuCyte analysis after 48 h of treatment with indicated drugs at the following IC50 cell viability concentrations (see ): azacitidine, 22.24 μM; decitabine, 10 μM; iBET151, 18.78 μM; JQ1, 5.26 μM; mivebresib, 3.09 μM; panobinostat, 0.278 μM; and TSA, 0.246 μM. (n = 2). (F) Western blot analysis of protein levels of WT and indicated mutants of FLAG-tagged RNF168 48 h after transient transfection. The samples are taken from parallel cells as used for reporter assays shown in .

Article Snippet: A549, H358, H460, and HBEC were authenticated (STR profiling) by the European Collection of Authenticated Cell Cultures in December 2018.

Techniques: Western Blot, Inhibition, Activation Assay, Transformation Assay, Mutagenesis, Expressing, Biomarker Discovery, Transfection