Journal: iScience

Article Title: Circ-Hdgfrp3 shuttles along neurites and is trapped in aggregates formed by ALS-associated mutant FUS

doi: 10.1016/j.isci.2021.103504

Figure Lengend Snippet: Key resources table

Article Snippet: N/A Mouse: mESCs carrying FUS P517L mutation in homozygosity Provided by Prof. Niel A. Shneider (Columbia University) N/A Mouse: Neuro-2a (N2a) ATCC cat#CCL-131™ Human: SK-N-BE cell lines expressing of the transgenes WT FUS and FUS-P525L ( Morlando et al., 2012 ) N/A Oligonucleotides DNA oligonucleotides for qPCR experiments used in this work, see Table S1 This paper N/A siRNAs for cell transfection used in this work: Dharmacon ON-TARGETplus targeting the circ-Hdgfrp3 BSJ (Sense sequence: 5'-CGG UGAAGGGAUUGAUGAAUU-3', Antisense sequence: 5'-UUCAUCAAUCCCUUCAC CGUU-3'); scramble RNA (Non-targeting Pool #D-001810-10-20) This paper N/A DNA oligonucleotides used to clone human circ-Hdgfrp3 into p-circ-3xF expression vector, see Table S2 This paper N/A Custom DNA circ-Hdgfrp3-specific probe for BaseScope™ This paper cat#703021 Negative control probe DapB for BaseScope™ ( Rossi et al., 2019 ) cat#701021 Custom DNA circ-ZNF609-specific probe for BaseScope™ ( Rossi et al., 2019 ) cat#708461 Recombinant DNA p-circ-3xF used for cloning human circ-Hdgfrp3 expression vector ( Legnini et al., 2017 ) N/A p-circ-Hdgfrp3 vector used for the expression of human circ-Hdgfrp3 This paper N/A Software and algorithms SDS Applied Biosystem 7500 Fast Real-Time PCR system software ThermoFisher Scientific N/A ImageJ software ( Schneider et al., 2012 ) https://imagej.nih.gov/ij/download.html MetaMorph® Microscopy Automation and Image Analysis Software Molecular Devices RRID:SCR_002368; https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref ImageJ Plot “Z-axis profiler” plugin DimiterProdanov;ZAxis_Profiler.java https://imagej.nih.gov/ij/plugins/dynamic-profiler/index.html ImageJ “3D viewer” plugin Benjamin Schmid;ImageJ_3D_Viewer.jar https://imagej.nih.gov/ij/plugins/3d-viewer/ ImageJ “JACoP (Just Another Colocalization Plugin)” plugin Fabrice P. Cordelieres, Institut Curie, Orsay (France)( Bolte and Cordelières., 2006 ) https://imagej.nih.gov/ij/plugins/track/jacop.html Open in a separate window Key resources table

Techniques: Transduction, Recombinant, Electron Microscopy, Modification, Knock-Out, Transfection, Clone Assay, Mutagenesis, Expressing, Sequencing, Plasmid Preparation, Negative Control, Software, Real-time Polymerase Chain Reaction, Microscopy

Journal: iScience

Article Title: The non-adrenergic imidazoline-1 receptor protein nischarin is a key regulator of astrocyte glutamate uptake

doi: 10.1016/j.isci.2022.104127

Figure Lengend Snippet: Nischarin mediates glutamate-dependent GLT-1 internalization in astrocytes (A) Surface biotinylation assay showing surface GLT-1 level in astrocytes transfected with GFP and GLT-1a-V5 or GFP-Nisch and GLT-1a-V5 following +/− 100μM glutamate treatment. One-way ANOVA, post hoc Dunnett’s multiple comparison test (n = 4 individual experiments). (B) Proximity ligation assay in DIV14 hippocampal culture. Increased red puncta per nuclei (DAPI stained (blue)) is indicative of increased direct interaction between Nischarin and GLT-1 in hippocampal culture. Glutamate treatment (100μM, 1h) significantly increased GLT-1-Nischarin interaction compared to control. One-way ANOVA, post hoc Tukey’s test (n = 3 individual preparations). (C) Schematic representation of GLT-1BBS construct bound to BTX conjugated Alexa 555 (BTX555). Astrocytes expressing GFP-Nisch and GLT-1aBBS were labeled using BTX555 and dual color live-structured illumination microscopy-monitored trafficking of GLT-1 following glutamate treatment. Merged kymographs of GFP-Nisch vesicle (green) and GLT-1 bound BTX-555 (red) reveal co-localized diagonal trajectory, representing moving vesicles. (D) Quantification of GFP-Nisch and GLT-1aBBS expressing astrocytes treated with 100μM glutamate for 0, 5, 30, and 60min showed increased colocalization between Nisch and GLT-1 compared to untreated controls. p values by unpaired t test, Mann Whitney test (n = 6-14).

Article Snippet: For measuring colocalization, the colocalization plugin of the Metamorph software was used.

Techniques: Surface Biotinylation Assay, Transfection, Comparison, Proximity Ligation Assay, Staining, Control, Construct, Expressing, Labeling, Microscopy, MANN-WHITNEY

Journal: PLoS ONE

Article Title: ADAMTS-1 Is Found in the Nuclei of Normal and Tumoral Breast Cells

doi: 10.1371/journal.pone.0165061

Figure Lengend Snippet: Immunofluorescence shows co-localization of ADAMTS-1 and nucleophosmin in MCF-10A, MCF-7 and MDA-MB-231 cell lines (A). MDA-MB-231 image used for colocalization analysis (B), with the white line used to generate the linescan plot (C). Linescan profiles display a clear overlap between ADAMTS-1 (Red) and nucleophosmin (Green). The colocalization color map is illustrated in C, where hot colors represent positive correlation (colocalization), and cold colors represent exclusion. Scatter plot (D) of ADAMTS-1 (red, x-axis) and nucleophosmin (green, y-axis) with the linear regression line. Points of scatterplot cluster around regression line. ADAMTS-1 (red), nucleophosmin (green) and nuclei (blue). Scale bar, 5 μm.

Article Snippet: Colocalization analysis was carried out by the Linescan tool (Metamorph Premier 7.6 software), and the Image J plugins JaCop, Colocalization colormap and Colocalization threshold.

Techniques: Immunofluorescence