Effective delivery of platelet miR-223 into A549 cells by P-MVs. (A) Comparison of the absolute levels of miR-223 in platelets and A549 cells. The level of miR-223 was assessed by qRT-PCR and normalized against the amount of total RNA. (B) Comparison of the absolute levels of miR-223 in P-MVs and A549 cells. The level of miR-223 was assessed by qRT-PCR and normalized against the amount of total RNA. (C) Flow chart of the experimental design. (D) Confocal microscopy image of the internalization of fluorescently labeled MVs into A549 cells. Platelets were labeled with DiI-C16 (red). After incubation for 4 h, the supernatants were collected and centrifuged to harvest P-MVs. The isolated P-MVs were re-suspended in RPMI 1640 medium supplemented with 10% FBS and incubated with A549 cells at 4°C or 37°C, respectively. After incubation for 2 h, A549 cells were washed, fixed, and observed under confocal microscopy. (E-F) Quantitative RT-PCR analysis of miR-223 (E) and pre-miR-223 (F) expression level in A549 cells treated without P-MVs (mock) or with P-MVs from various sources for 24 h. Data are presented as the mean ± SEM of five independent experiments (*, P < 0.05, **, P < 0.01,***, P < 0.001).