Fluorescent reporter protein remains primarily in the supernatant in assays for EV-to-cell transfer.

(A) Cartoon / description of experiments to probe uptake of EVs by U87MG cells, examining small EV uptake +/- bafilomycin A1 (BafA1) (B) or comparing small EV uptake to larger EV uptake (D). LTR = long terminal repeat, WPRE = woodchuck hepatitis virus post-transcriptional regulatory element, EF1a = translation elongation factor 1a promoter (B), U87MG cells were seeded and grown 24 hr on 96-well plates at which point the media was replaced with serum free media (no additional serum was added for the remainder of the experiment), and wells of serum-free media were aliquoted to use as a cell-free culture control. The plate was incubated for 4 hr, and cells and cell-free media wells were treated with 200 nM bafilomycin A1 (BafA1) or a vehicle control (+/-). The plate was returned to the incubator for 1 hr, then EVs (small and large EVs, see “Materials and Methods”) were added to a concentration of 20 μg/ml. Two wells were used for each condition (technical replicates). At 1 hr, 24 hr, and 96 hr, samples were collected from the conditioned media (cell supernatant, S), and 15% of the total supernatant was resuspended with 5X SDS-PAGE loading dye to lyse. The bottom of the wells containing adhered cells (“cells” = C) were washed 2X with PBS, and cells were removed by suspending in a volume of 1X SDS-PAGE loading buffer equivalent to samples prepared for the supernatant. Lysates of 15% of the supernatant (S) and 100% of the well bottom (“cells” = C), for the two technical replicates, were resolved by SDS-PAGE and were immunoblotted as indicated. (C) HA-TagBFP-HVR-CAAX signals were quantitated from the immunoblot (B) and from the replicate not shown in (B), subtracting background from recorded intensities and normalizing the “cell” signals (multiplying by 0.15) for comparison to background subtracted supernatant signals (which were loaded at 15%). Cell co-culture signals for supernatant or “cell” were then divided by the total signal detected in cell-free co-culture with EVs (background subtracted supernatant signal summed with 15% of background-subtracted “cell” signal) to give a percentage. This percentage represents the amount of the fusion protein remaining in the media (supernatant) or associated with cells (either on the plate, on the cell surface, or inside of cells) when co-cultured with cells as compared to the total amounts of fusion protein detectable at the time point analyzed for cell-free culture. Scatter plot of signals in (B) showing percentage of total Tag-BFP fusion (total detected in cell-free co-culture) for cell and supernatant fractions at 1 hr and 24 hr time points for this experiment. (D) SDS-PAGE of lysates of supernatant or “cell” fractions from cells or media cultured with EVs for time points as in (B)but large EVs (P16,500xg) or small EVs (P110,000xg) fractions were separately incubated with cells or media, and BafA1 treatment was not analyzed. (E) Calculations as in (C) for P110,000xg Tag-BFP fusion uptake at 1 hr and 24 hr (n = 3 independent experiments). The lines show the position of the means.