Additional file 1 of miR-210-3p enriched extracellular vesicles from hypoxic neuroblastoma cells stimulate migration and invasion of target cells
Creators: name: Fusco, Pina
name: Fietta, Anna
name: Esposito, Maria Rosaria
name: Zanella, Luca
name: Micheli, Sara
name: Bastianello, Angelica
name: Bova, Lorenzo
name: Borile, Giulia
name: Germano, Giuseppe
name: Cimetta, Elisa
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Marine Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. EVs characterization. A. Diameters of isolated EVs measured from TEM analysis. B. Representative EVs size distribution and concentration measured by qNano analysis. C. Representative EVs size distribution and concentration measured by nanoparticle tracking analysis. D. Background corrected APC median signal intensities for all 39 capture bead populations of the EVs surface from SK-N-AS and SK-N-DZ cells cultured in normoxia (grey bar), hypoxia (red bar), and reoxygenation (blue bar) conditions. Bars represent the mean ±SEM from at least three independent experiments. Figure S2. Uptake of NB-EVs. All experiments are performed on SK-N-AS cells treated with EVs from SK-N-AS cells cultured in normoxic, hypoxic and reoxygenation conditions. A. Uptake of NB-EVs by SKN-AS cells. Cells were cultured for 8 and 24 h in the presence of DiO-labelled EVs. Cells were stained with the Alexa Fluor 488 phalloidin (red) and DAPI (blue). Magnification 40X. Scale bar, 100 μm. B. Fluorescence intensity was evaluated in five random fields in sections using Image J. Graph represent values from at least n=3 independent experiments. *p&lt;0.05, **p&lt;0.01 and ****p&lt;0.0001 vs normoxia. Figure S3. Heatmap shows a panel of 250 miRNAs with fluorescence intensity above the detection threshold (AU &gt; 4.71) in each normoxia, hypoxia and reoxygenation condition. Figure S4. The workflow of miRNA filtering adopted comparing oxygenation condition between two cell lines is shown. Figure S5. Characterization of EVs from NB cells after transient transfection with miRNA-210-3p mimic and inhibitor. A. Transmission electron microscopy images of EVs isolated from cells transfected with miR210-inhibitor and miR210-mimic cultured in normoxic conditions. B. Validation of EVs markers expression by western blot analysis. C. Representative EVs size distribution and concentration measured by nanoparticle tracking analysis. Figure S6. EVs derived from transfected cells affected migration and invasiveness of SK-N-AS and SK-N-DZ cells. A. Representative images of wound healing assay performed on cells treated with EVs from transfected SK-N-AS and SK-N-DZ cells with miR-210-3p mimic; images were acquired at the time of EVs addition (T0) and 48 hours after treatment (T48H). Magnification 10X. Scale bar, 100 μm. Wound closure areas and cell velocity (μm/h) were quantified with Image J. Values are expressed as mean}SEM from 3 independent experiments. B Quantification of cells invaded toward the Matrigel layer (average of 5 picture fields at 10x magnification) were quantified with Image J. Values are means } SEM. C. Representative images of wound healing assay performed on cells treated with EVs from transfected SK-N-AS and SK-NDZ cells with miR-210-3p inhibitor; images were acquired at the time of EVs addition (T0) and 48 hours after treatment (T48H). Magnification 10X. Scale bar, 100 μm. Wound closure areas and cell velocity (μm/h) were quantified with Image J. Values are expressed as mean}SEM from 3 independent experiments. D. Quantification of cells migrated toward the Matrigel layer (average of 5 picture fields at 10x magnification) were quantified with Image J. Values are means} SEM.