Fig. 3. Cell death evaluation by PI and Annexin-5.

Cell death was evaluated by PI assay, on Hepa-RG cells, untreated (CTR) (A), and after incubation with NAFLD-Exo (B) and Healthy-Exo samples (C). Experiments were repeated three times and the PI% intensity was reported in (D). **P < 0.001. Western blotting assay was performed for Annexin V. Negative controls were untreated cells normalized using GAPDH in the same way as the cells treated with NAFLD-Exo and Healthy-Exo samples (E). In the histogram, semi-quantitative evaluation, performed by means of video-densitometry, of the relative expression levels of the Annexin-5 (F, average of three experiments). For all the experiments, the exosomes concentration was fixed at 20 µg/µL in terms of total protein content.