Fig 3: A subpopulation of small EVs is highly enriched in RNA and is regulated by VAP-A.

(A) Representative immunoblot of different EV cargo and marker proteins (Ago2, flotillin-1, hnRNPA2B1, LC3B, VAP-A, Alix, syntenin, TSG101 and CD63) shows segregation in fractions 3 and 5 of “light” and “dense” EVs purified from control DKs-8 cells. Representative of n=3 independent experiments.

(B) NTA traces for “light” and “dense” EVs purified from control (Sc) and VAP-A KD2 (KD) DKs-8 cells. Median values from three independent experiments were plotted.

(C) EV concentrations were calculated from NTA data and plotted from three independent experiments.

(D and E). Representative TEM images of “light” and “dense” EVs purified from control (Sc) and VAP-A KD2 (KD) DKs-8 cells are shown. Graphs show size of the “light” and “dense” EVs calculated from a total of 150 vesicles per condition from three independent experiments.

(F and G) Total RNA quantity measured by NanoDrop (A260) or Qubit (fluorescence) per “light” and “dense” small EVs isolated from control and KD cells. Data from three independent experiments. Note the enrichment of RNA in dense EVs compared to light EVs, regardless of the method of measurement.

(H) Fold change values of specific miRNAs in control and KD light and dense small EVs show VAP-A KD selectively affects the dense EV population. Data from three independent experiments and candidate miRNA Ct values were normalized by respective U6 values which is unchanged.

(I and J) Specific miRNAs (let-7a, miR-100, miR-320a and U6) are present inside light and dense small EVs, as they are only susceptible to RNase treatment in the presence of Triton-X-100 (TX100). Data from three independent experiments.

Data plotted as mean +/− S.E.M. Scatter plots indicate median and interquartile range.

*p<0.05, **p<0.01, ***p<0.001. ns, not significant.