Fig. 2.

Low-dose 2 and 4-month exposure to OTA induces sEV secretion in the conditioned media of NRK52E cells. A Chromatograms showing the size (x-axis) and concentration (y-axis) of exosomes isolated from 48 h (upper panel), 2-months (middle panel) and 4-months (lower panel) of OTA-exposed NRK52E cells. B Bar diagram representing the concentration of isolated EVs. C Characterization of transmission electron microscopy (TEM) representing the spherical shape and size (nm) of EVs isolated from OTA-treated cells (upper panel for 48 h, middle panel for 2 months, and lower panel for 4 months), original magnification X67000, 80 kV, scale bar 200 nm. D-E Representative images of immunoblotting for sEVs positive markers, CD63, CD9, flotillin-1 expression D and sEVs negative markers, calnexin and GM130 E in EVs isolated from both 48 h, 2 and 4-month OTA-exposed and control NRK52E cells. EVs were isolated from equal volumes of conditioned media, and equal amount of EV proteins was used for immunoblotting. F Immunogold-TEM images further validate the expression of CD9 protein on the surface of four-month OTA-induced sEVs. The upper lane shows X150000 magnification with a 100 nm scale bar, and the lower lane shows a zoomed-in view. The red arrow represents the CD9 expression on the surface of sEVs. 48 h = 48 h OTA-treated NRK52E cells, 2 M = two-month OTA-treated NRK52E cells, 4 M = four-month OTA-treated NRK52E cells, Control = vehicle-treated NRK52E cells, LD = 250 nM OTA-treated NRK52E cells, and HD = 500 nM OTA-treated NRK52E cells, WCL = whole cell lysate of NRK52E cells