Figure 5. Bystander autophagy induced by the exosomal miR-7-5p in recipient BEP2D cells.

Panel A: Observation of autophagy induced by exosomes. BEP2D cells were irradiated with 2 Gy of ⁶⁰Co γ-rays. The exosomes were harvested 4 hr post-irradiation, and co-treated the non-irradiated cells with pEGFP-C1-LC3 vector in the presence (IR-exosome + miR-7-5p inhibitor) or absence (IR-exosome) of miR-7-5p inhibitor. The autophagic vacuoles were determined by fluorescent staining. Nuclei were stained blue by DAPI, while exosomes were stained red, and green was LC3-GFP. The effect of exosomes from the non-irradiated cells were also performed as the control (Con-exosome). Panel B: The number of autophagosomes (LC3 punctium) in the exosomes-treated BEP2D cells was counted in 20 randomly selected positive cells (red and green). *p < 0.01: IR-exosome vs Con-exosome. ^#p < 0.01: IR-exosome + miR-7-5p inhibitor vs IR-exosome. Panel C: Western blotting analysis of the exosomal proteins Tsg101, Alix, CD63 in BEP2D cells and the exosomes. Panel D: Observation of autophagy induced by the conditional medium from irradiated cells. BEP2D cells were irradiated with 2 Gy of ⁶⁰Co γ-rays. The conditional medium was collected 4 hr post-irradiation. After removing cellular debris by centrifugation, the exosomes-containing conditional medium (IR-medium) and exosome-free medium (IR-medium-exosome free) were used to treat the non-irradiated BEP2D cells. The exosomes-free medium was prepared by further super-speed centrifuging the conditional medium to remove the exosomes at 100,000 g for 70 min. Panel E: The number of autophagosomes (LC3 punctium) in the medium-treated BEP2D cells was counted in 20 randomly selected positive cells (green). *p < 0.01 as compared with untreated cells. ^#p < 0.01 as compared with the cells treated with the medium from irradiated cells.