Figure 2.

Megakaryocytes produced pro-survival exosomes. K562 cells were treated with PMA (10 nM) in the absence or presence of dipyridamole (10 µM) or aspirin (100 µg/mL) for 4 days. Equal amounts of cultured media were subjected to exosome isolation. (A,B) The obtained exosomes were subjected to flowcytometric analysis for the measurement of CD9 positive particles. (C) The obtained exosomes were subjected to the measurement of protein content. (D) The obtained exosomes were subjected to Western blot analysis with indicated antibodies. Representative blots of four independent experiments are shown. (E) Various concentrations of exosomes based on protein contents were added to T24 cells (96-well) for 48 h. Cell viability was measured by the MTS reduction assay. T24 cells were treated with doxorubicin (0 and 2 µM) in the absence or presence of the obtained exosomes (5 µg/mL), dipyridamole (10 µM), or aspirin (100 µg/mL) for 48 h. (F) Cells were harvested and subjected to flowcytometric analysis for the measurement of SubG1 population. (G) Proteins were extracted and subjected to enzymatic assay for the measurement of caspase 3 activity. T24 cells were treated with doxorubicin (0 and 2 µM) in the absence or presence of high-mobility group box 1 (HMGB1) (5 µg/mL) for 48 h. (H) Cells were harvested and subjected to flowcytometric analysis for the measurement of SubG1 population. (I) Proteins were extracted and subjected to enzymatic assay for the measurement of caspase 3 activity. * p < 0.05 vs. untreated group, # p < 0.05 vs. exosome (vehicle, 5 µg/mL) or doxorubicin (2 µM), and % p < 0.05 vs. doxorubicin (2 µM)/exosome (vehicle, 5 µg/mL), n = 4.