6533b7d9fe1ef96bd126d6d1

RESEARCH PRODUCT

Additional file 1: of CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA

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(a) Characterization of isolated exosomes. Left panel: DSL for exosomes released by SKHep Middle panel: Western blot forTsg101 and HSC70 in SkHep cells and their relative exosomes. Right panel: Confocal microscopy analysis on HUVECs treated for 1, 3 and 6 hours with 5 mg/ml of SKHep-derived exosomes. HUVECs were stained with phalloidin Alexa Fluor (green), nuclear counterstaining was performed using DAPI (blue), exosomes were labelled with PKH26 (red). (b) Target analysis. Real time-PCR analysis on HUVECs treated for 18 h with 5 mg/ml of SkHep-derived exosomes. Normalized for b-actin the DDct were indicated as fold of induction respect to control (untreated cells). *p<0.05. (c) Tubulogenesis of HUVECs after exosomes treatment. Matrigel assay performed on HUVECs cells after 18 hour of 5 mg/ml SkHep-derived exosomes. Left panel: phase contrast, magnification 20x. Right panel: quantification of matrigel assay expresses as length of cable as arbitrary unit **p<0.01. (d) Adhesion assay of SkHep cells on HUVECs. Left panel: phase contrast, magnification 10X. Right Panel: quantification of Huh7 or SKHep cells adherent on HUVECs, performed by counting the number of CD90+adherent cells (violet) per field ***p<0.001. (e) Analysis on H19 expression in exosomes. Real time-PCR analysis for H19 expression performed on SkHep-derived exosomes respect to Huh7 derived exosomes. Normalized for b-actin the DDct were indicated as fold of induction. **p<0.01 (f) Comparison of H19 expression in HUVECs after exosomes treatment. Real time-PCR analysis for H19 expression on HUVECs treated for 18 h with 5 mg/ml of SkHep or Huh7 exosomes. Normalized for b-actin the DDct were indicated as fold of induction respect to control (untreated cells). (DOCX 855 kb)