Fig. 3

EV subpopulations enhance cell-to-cell contact, which is abolished by ICAM-1 or CD45 siRNA. a Fluorescent Microscopy was used to track BODIPY™ 493/503 labeled EVs (2 k at 1: 6,200 cell to EV; 10 k at 1: 20,000; 100 k at 1: 8,200, and CEM at 1: 8,866) added to CEM recipient cells. Analysis was performed 24 h post-treatment and quantified total and aggregated cells. Scale bar (100 μM). b Cell viability of recipient cells (CEM; 5 × 10⁵ cells/mL, in biological triplicates) incubated with HTLV-1 2 k, 10 k, 100 k, and total EV populations (CEM and HUT102). c Cells and corresponding total EVs (CEM and HUT102), d as well as subpopulations (2 k, 10 k and 100 k EVs), were analyzed via Western blot for cell surface proteins (CD45, CD43, ICAM-1, LFA-1), HTLV-1 proteins (gp46, and p19), EVs (CD63), and Actin. e The 10 k and 100 k EVs were isolated (single 100,000 × g spin) from HUT102 cells transfected cells with siRNA against ICAM-1, CD45, or scramble (for 3 days). All samples were labeled with BODIPY and then incubated with CEM cells at a ratio of 1:10,000 cell to EV for 3 days. Total HUT102 EVs was used as positive control for enhanced cell-to-cell contact and CEM cells alone or with scrambled siRNA as a negative control. The images were taken by Fluorescent Microscopy. f HTLV-1 donor cells (HUT102) were irradiated (IR; 10 Gy) to induce cell cycle arrest and treat recipient cells (CEM) in fresh exo-free media for 4 days. Western blot analysis was performed for p19 and Actin. Statistical analyses were performed using two-tailed Student’s t test, “*” for p ≤ 0.05 and “**” for p ≤ 0.01