FIGURE 2.

Inhibition of NKG2D expression by exosomal MICA/B. (A) IMR-32 cells were treated with 2 μM MLN8237 or 0.5 μM DOX for 72 h. No treatment was used as a control, and exosomes were isolated by ultracentrifugation. Western blot was used to evaluate CD63 and TSG101 levels in whole cell lysates of the cells or isolated exosomes from supernatants. (B) Representative EM image of exosomes and showing the typical cup-shaped morphology. Scale bar, 100 nm. (C) Analysis of particle size distribution and concentration in exosomes. (D) IMR-32 cells were treated with 2 μM MLN8237 for 72 h, no treatment was used as a control. The expression of MICA/B in IMR-32 cells and exosomes were determined by flow cytometry, and exosomes were pretreated by coated latex microbeads. (E) Normal human peripheral blood NK cells were used as a control. Exosomes isolated from untreated IMR-32 cells, and MLN8237-treated cells were co-cultured with NK cells for 24 h. Co-cultures were spiked with the MICA/B antibody, and NKG2D expression was detected by flow cytometry.