Journal: PLoS ONE

Article Title: Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

doi: 10.1371/journal.pone.0060152

Figure Lengend Snippet: (A) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 4 h and processed for electron microscopy. Images show neuronal cell bodies at the level of the nucleus, demonstrating vacuolation in the presence of YM-201636. Grid size = 1 µm square. (B-D) Electron microscopic analysis of the vacuole area as a percentage of total cytoplasmic area (B) and the number of vacuoles per cell (C) shows a significant increase in vacuole size and number following 4 h treatment with 1 µM YM-201636, **p<0.01, ***p<0.001. (D) Histogram of number of vacuoles relative to their size (representative experiment), n = 12 (DMSO) or 11 (YM-201636) cells. (E) Examples of vacuole phenotypes detected in primary hippocampal neurons treated with 1 µM YM-201636 for 4 h. (F) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 4 h or 18 h and immunolabelled for LAMP1, EEA1 and ß3-tubulin. 3D projections are shown. Scale bar = 10 µm.

Article Snippet: Antibodies were obtained from the following sources: rabbit anti-LC3 (Novus Biologicals, NB100-2331), mouse anti-ß-actin (Sigma, S0644), mouse anti-ßIII tubulin (Covance, MMS-435P), rabbit anti-EEA1 (#2411) and rabbit anti-cleaved caspase-3 (#9661) (Cell Signaling Technology), mouse anti-LAMP1/LY1C6 (Sapphire Bioscience, #120-13523), rat anti-LAMP1/CD107a (#553792), and mouse anti-GM130 (#610822) (BD Biosciences).

Techniques: Electron Microscopy

Journal: PLoS ONE

Article Title: Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

doi: 10.1371/journal.pone.0060152

Figure Lengend Snippet: Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 for 4 h, then supplemented with 5 µg/ml WGA-Alexa555 for the final 5 min (A,B) or 30 min (C,D). Cells were fixed, immunolabelled for EEA1 and LAMP1, and imaged by confocal microscopy. (B,D) The intensity of WGA-Alexa555 per µm 2 in the cell body and the total area of the cell body were determined in the YM-201636–treated cells relative to DMSO. (E,F) Primary hippocampal neurons were treated with DMSO or 1 µM YM-201636 in the presence of 5 µg/ml WGA-Alexa555 for the full 4 h. Cells were fixed, immunolabelled for GM130 and imaged by confocal microscopy. (F) The amount of WGA-Alexa555 fluorescent intensity in the cell body/µm 2 of the YM-2016363 treated cells was determined relative to DMSO. The area of the cell bodies analysed was also determined (mean ± SEM, n = 3 independent experiments, 9–28 cells per experiment). Significances relative to DMSO **p<0.01. Scale bar = 10 µm.

Article Snippet: Antibodies were obtained from the following sources: rabbit anti-LC3 (Novus Biologicals, NB100-2331), mouse anti-ß-actin (Sigma, S0644), mouse anti-ßIII tubulin (Covance, MMS-435P), rabbit anti-EEA1 (#2411) and rabbit anti-cleaved caspase-3 (#9661) (Cell Signaling Technology), mouse anti-LAMP1/LY1C6 (Sapphire Bioscience, #120-13523), rat anti-LAMP1/CD107a (#553792), and mouse anti-GM130 (#610822) (BD Biosciences).

Techniques: Confocal Microscopy

Journal: Theranostics

Article Title: Mesenchymal Stromal/stem Cell-derived Extracellular Vesicles Promote Human Cartilage Regeneration In Vitro

doi: 10.7150/thno.20746

Figure Lengend Snippet: BMMSC-EVs are taken up by chondrocytes derived from OA patients. CFSE labelled EVs derived from TERT-bone marrow MSC were incubated for 30 min with OA chondrocytes and the uptake of EVs was analyzed by confocal microscopy. Left panel: Representative images of co-localization analyses of CFSE labelled (green) BMMSC-EVs with late endosomal marker LAMP1 (red) in OA chondrocytes are shown. Arrow heads indicate the co-localization (yellow) of BMMSC-EVs with LAMP1. Right panel: Representative images of co-localization analyses of CFSE labelled (green) BMMSC-EVs with early endosomal marker EEA1 (red) in OA chondrocytes is shown. Images are representative of 3 independent experiments. Scale bar is 10 µm.

Article Snippet: For BMMSC-EV uptake analysis, fixed OA chondrocytes were incubated with 2% BSA for 30 min followed by 1 h incubation with a rabbit anti-EEA1 antibody (1:200; Cell Signaling) and mouse anti-LAMP-1 (1:400, BD Pharmigen) followed by subsequent incubation with a donkey-anti-rabbit antibody labelled with DyLight 549 (Jackson) and a goat-anti-mouse antibody labelled with DyLight 649 (Jackson), respectively.

Techniques: Derivative Assay, Incubation, Confocal Microscopy, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Metastatic state of parent cells influences the uptake and functionality of prostate cancer cell-derived extracellular vesicles

doi: 10.1080/20013078.2017.1354645

Figure Lengend Snippet: Localisation of the EV-associated DiIC 18 (5)-DS label in prostate cells. ( a ) Cells were incubated for 16 h with PC-3 and PNT2 DiIC 18 (5)-DS labelled EVs (DiIC 18 -EVs) (pseudo-coloured green). Cells were immunostained with antibodies against CD44 for the localisation of plasma membrane (PM), EEA1 for early endosomes (EE), Lamp1 for lysosomes (LYS), and GM130 for Golgi (red), and imaged by confocal microscopy. Scale bars 10 µm. Insets show a zoom into the organelle. ( b ) The percentage of the DiIC 18 dye co-localisation with each antibody was quantified by Imaris software ( n = 42–110 cells). Error bars represent the mean ± SEM of 8–36 image frames each, * p < 0.05 unpaired t-test.

Article Snippet: Samples were stained at 4°C overnight with anti-CD44 (1:200, gift from Dr Jalkanen, Turku, Finland), anti-Lamp1 (H4A3, 1:100, IOWA University, Developmental Studies, USA), polyclonal anti-GM130 (1:100) or anti-EEA1 (1:100) (Cell Signaling, Danvers, MA, USA).

Techniques: Incubation, Confocal Microscopy, Software

Journal: Journal of Nanobiotechnology

Article Title: Quantification of the internalization patterns of superparamagnetic iron oxide nanoparticles with opposite charge

doi: 10.1186/1477-3155-10-28

Figure Lengend Snippet: Intracellular localization of SPIONs. Cells were incubated concomitantly with both SPIONs (γ-Fe 2 O 3 -PEI-FITC and γ-Fe 2 O 3 -PMA-Dy636) at a final concentration of 1 μg/ml [Fe] for time periods of 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. Cells were then stained with wheat germ agglutinin, for EEA1 and for LAMP-1 to visualize the Plasma Membrane (white), the Endosomes (yellow) and the Lysosomes (red), respectively. Fluorescence images were recorded with a LSM. Additionally, the same experiments were performed with cells incubated with each NP system (see ).

Article Snippet: Lysosomes were stained using monoclonal mouse anti-human LAMP1/CD107a antibodies (Developmental Studies Hybridoma Bank), while early endosomes were labeled with polyclonal rabbit anti-human EEA1 immunoglobulin (Cell Signaling).

Techniques: Incubation, Concentration Assay, Staining, Fluorescence

Journal: Emerging Microbes & Infections

Article Title: GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus

doi: 10.1080/22221751.2019.1677446

Figure Lengend Snippet: GILT expression is upregulated by IFN-γ in lung cancer cells and has a lysosomal distribution. A549 and MRC-5 cells were treated with the indicated concentrations of IFN-α or IFN-γ for 12 h. (A) The levels of intracellular GILT and IFITM proteins were determined by Western blot assay. β-actin served as a loading control. (B) The localization of GILT in MRC-5 cells treated with IFN-γ was detected by immunofluorescent staining with anti-GILT polyclonal antibody (green). EEA1, Rab9 or LAMP1 were visualized by immunofluorescent staining with respective antibodies (red). Cell nuclei were stained blue with DAPI.

Article Snippet: To visualize the subcellular localization of wild-type and mutant GILT proteins, A549 or TRex 293 cells were first fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 for 10 min. After incubation in the blocking solution (1×PBS with 3% BSA and 5% FBS) for 1 h, cells were stained with a mouse monoclonal antibody (G-11, SANTA CRUZ sc-393507A) or rabbit polyclonal antibody recognizing GILT protein (sc-393507A) together with a Rabbit derived monoclonal antibody against EEA1 (Cell Signaling, 2411), Rab9 (Cell Signaling, 5118s) or mouse derived LAMP1 monoclonal antibody (Cell Signaling, 15665), respectively.

Techniques: Expressing, Western Blot, Staining