Figure 2. Ethanol treatment increases total number of EVs in a dose- and time-dependent manner in CAL27 cells.

CAL27 cells were cultured, and numbers of EVs, exosomes (size <150 nm), and MVs (size >150 nm) were quantified in cell-free supernatant by NanoSight. (A) CAL27 cells were exposed to various dose of ethanol (25 mM, 50 mM, 100 mM), LPS (10 ng, 100 ng) or left untreated for 24 h, and the frequency of vesicles was determined. (B) CAL27 cells were exposed to various dose of ethanol (25 mM, 50 mM, 100 mM), LPS (10 ng, 100 ng), or left untreated for 48 h, and the frequency of vesicles was determined. (C) CAL27 cells were exposed to various dose of ethanol (25 mM, 50 mM, 100 mM), LPS (10 ng, 100 ng), or left untreated for 72 h, and the frequency of vesicles was determined. (D) Kinetic of EV production after challenge with 50 mM ethanol was determined at each condition by enumerating the number of EVs in the cell supernatants. (E) Size distribution of EVs produced by CAL27 cells in 24 h in the unchallenged condition. (F) Size distribution of EVs produced by CAL27 cells challenged by 25 mM ethanol in 24 h. (G) Size distribution of EVs produced by CAL27 cells challenged by 10 ng LPS in 24 h. Nanosight device was calibrated with 100 nm polystyrene beads before each set of measurements. All measurements were done in triplicate from 6 independent samples in each group. (H) Scanning electron microscopy image of untreated CAL27 cells are shown (×3000 magnification). The blown-up image of the selected region (×7500 magnification) in the cancer cells shows the shedding of EVs on the surface of the cells. (I) EVs originated from CAL27 cells carry small noncoding RNAs in size range of miRNAs. Figure is the representation of EV-encapsulated small RNA profile obtained from 4 independent sample preparations. (^*indicates p < 0.05 versus control).