MyD88^L265Ptransferred by the EVs forms aggregates with endogenous MyD88 in the recipient cells. (A) EV_L265P-CFP (cyan) were isolated and added (5 μg/mL) to the HEK293 cells for 16 hours. Cell membranes were dyed with CT-B Alexa 647 (magenta). (B,D) HEK293 and HEK293 MyD88^KO cells were transfected with MyD88^wt-YFP (magenta) or (C) left untreated. After 4 hours, EV_L265P-CFP (cyan) or EV_L265P-YFP (magenta) (both 5 μg/mL) were added for 16 hours. Confocal imaging was performed. Bar for all images, 10 μm. (E) HEK293 and HEK293 MyD88^KO cells expressing luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated with EV_L265P (12 μg/mL) for 24 hours. Negative controls are transfected but unstimulated cells. Dual luciferase test for NF-κB activity was performed.