Fig. 4

Functional Effects of HTLV-1 EVs on Angiogenesis and Inflammation. a An angiogenesis assay in technical and biological triplicate was used to determine the effect of distinct HTLV-1 EVs (2 k, 10 k, and 100 k) on tubular formation in a co-culture of mesenchymal stem cells (MSCs) and aortic endothelial cells (AEC). EV-treated cells (1:2000 recipient cell to EV ratio). Positive control cells received complete, undiluted medium. Additional dose of EV treatment was given to the cells at day 4. Representative images were taken at days 3 and 6 showing tubular formation in response to the indicated treatment. b The image processing software WIMASIS was used to calculate the percentage of area covered by tubules (n = 3) on day 3 and day 6. c RT-qPCR results showing env RNA copy numbers of mesenchymal stem cells treated with CEM EVs (control) and different populations of HTLV-1 EVs: 2 k (left panel), 10 k (middle panel), and 100 k (right panel). A set of dashed black vertical lines (---) were used to indicate baseline env RNA copy numbers. A set of dashed red vertical lines ( ) were used to indicate the levels of starting material, suggestive of the minimum env RNA copy numbers necessary for EVs to increase vial spread in MSc. d Western blot analysis for core histones (H3, H2A, H2B, and H4), linker histone (H1) and actin in HUT102 EVs (2 k, 10 k, and 100 k). e GAPDH DNA levels (representative of nucleosomes) in 2 k, 10 k, and 100 k HUT102 EVs treated with proteinase K and DNase/RNase were evaluated by was quantitated by q-PCR. A two-tailed student t-test was used to evaluate statistical significance with “**” for p-values ≤ 0.01, indicating the level of significance relative to untreated (Control) samples