Fig. 5

Higher uptake of SFN-F-EXOs by HL-1cardiomyocytes is related to increased histone acetylation. A-B HL-1 cardiomyocytes were seeded in an 8-well chamber slide and treated with or without 100 nM AngII for 12 h. F-EXOs (75 μg/mL) or SFN-F-EXOs (75 μg/mL) were added to each well, and cells were incubated for further 12 h. PBS alone was added as a control. HL-1 were fixed, permeabilized and immunoprobed using an antibody against AcH4. Signal was detected by fluorescent microscopy (20X magnification); representative pictures are shown (A). Fluorescence intensity was normalized for cell area; data are presented as mean ± SEM of three independent experiments, each one analyzed on at least five not-overlapping fields (B). *p < 0.05. C-D Maspin levels in exosomes released from untreated and treated (SFN) NIH/3t3 and in exosome-free culture medium were measured by Western blotting. A representative blot and Ponceau S staining are shown. (C). Densitometric data were normalized over Ponceau staining, expressed as relative to the control group (Not treated, 0 μM SFN) and represent the mean ± SEM of three independent experiments (D). E–F In order to evaluate exosomes uptake by cardiomyocytes, HL-1 cardiomyocytes were seeded in an 8-well chamber slide and treated with 100 nM AngII for 12 h, DiA-labelled F-EXOs (100 μg) and SFN-F-EXOs (100 μg) were added to each well. After 3 h, cells were fixed and glass slides mounted in the presence of DAPI (nuclei, blue). Exosome uptake was detected by fluorescent microscopy (20X magnification); representative pictures are shown (E). Uptake was quantified as fluorescence intensity of DiA (green, representing the fluorescently-labelled exosomes)/cell surface area. Data are presented as mean ± SEM of three independent experiments, each one analyzed on at least five not-overlapping fields (F). *p < 0.05, ***p < 0.001, ****p < 0.0001