Fig. 4

SFN-F-EXOs reduce AngII-induced oxidative stress and apoptosis of murine HL-1 cardiomyocytes. 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. A-B Superoxide (O2-) generation was detected by DHE staining. Cells were fixed and signal was detected by fluorescent microscopy (20X magnification); a representative picture is shown for each treatment (A). Superoxide levels are shown as mean ± SEM of three independent experiments, each one analyzed on at least five not-overlapping fields (B). **p < 0.01, ***p < 0.001, ****p < 0.0001. C-D In order to evaluate apoptosis, procaspase-3 and cleaved caspase-3 levels were measured by Western blotting on HL-1 cells after F-EXO treatment. HL-1 cardiomyocytes were seeded in 6-well plates 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 before cell lysis and blotting. PBS alone was employed as a control. A representative blot is shown for each protein (C). Protein levels were normalized for the loading control (β-tubulin). Relative levels of cleaved caspase-3 to procaspase-3 are shown in the graph as mean ± SEM of 3 independent experiments (D). *p < 0.05, **p < 0.01