Fig. 2: pTDEs induce cellular ROS generation, DNA damage, and G2/M arrest, resulting in the inhibition of proliferation in recipient cells.

a Recipient cell viability after tumor-derived EV (TDE) treatment (0.1 µg/ml) was measured by MTT assays (n = 8). b The proliferation rate was manually measured by cell counting in the PBS and TDE treatment groups. pTDEs decreased cell viability and proliferation. c Cell cycle analysis of exosome-treated cells was performed using flow cytometry. pTDEs induced an increase in the proportion of cells in G2/M phase. d Representative images showing cellular ROS levels. Intracellular oxidative stress was measured with the fluorescent ROS probe 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA). A brighter green fluorescence indicates high ROS. EV-treated cells were stained with H2DCFDA to measure intracellular ROS. Scale bar, 170 μm. e For measurement of damaged DNA, γ-H2A.X (green) and DAPI (blue) were stained in the EV-treated CHMm cells. The white arrow indicates damaged DNA, which colocalized with γ-H2A.X and DAPI. Scale bar, 10 μm. f Western blot for intracellular γ-H2A.X, γ-H2A.X increased with pTDE treatment. g Tube formation assays of HUVECs to detect the angiogenic potential of pTDEs. HUVECs were seeded on Matrigel, and the exosomes were treated for 24 h. Representative images showing that pTDEs inhibited tube formation compared with that of the controls. Bottom, microscopy images were analyzed by the ImageJ plugin Angiogenesis Analyzer. The quantification of the mean mesh size is presented as the mean ± SEM. Two-way ANOVA and Tukey’s HSD test were used to compare groups; **P < 0.01, ***P < 0.001, and ns not significant. The results are presented as the mean ± SD.