Figure 2.

Buoyant density gradient ultracentrifugation demonstrates the presence of Long Interspersed Element-1(LINE-1) mRNA and ORF1p in high- and low-density extracellular vesicles (EVs). EVs from H460 cells stimulated with 1 μM BaP were subjected to iodixanol buoyant density gradient ultracentrifugation to examine the presence of LINE-1 mRNA and protein within EVs and their distribution across EV populations. (A) Mean densities of 1 mL fractions from three gradient preparations. Diagram showing how EVs rise to the gradient level equivalent to their density during the course of centrifugation. (B) Western blot of EV proteins present in density gradient fractions 1–12. ALIX and Flotillin-1 are proteins enriched in EVs. ORF1p, which often appears in multimers in SDS-PAGE, is visible as a putative dimer within EVs. Cell lysate is shown as a positive control, EV-free media (EFM) as a negative control. (C) Western blots from two additional independent gradients. (D) mRNA in gradient fractions. mRNA was quantified by One-Step RT-qPCR. Amplicons were expressed as fold-change from the Reverse Transcriptase Control (LINE-1, top) or Non-Template Control (β-Actin, bottom). For LINE-1, the minimum, maximum, and mean are shown in box plot. β-Actin, mean and SEM shown. N = 3–4.