Dapi solution

Around 25,000 HeLa cells were seeded on a 24-well plate containing sterile 12 mm microscopic coverslips (ThermoFisher Scientific, Waltham, MA, USA). After 24 h, normal growth media was replaced with FBS18 media. Thirty microliters of HEK-CD63-GFP TSU and PSU sEV fractions (F1–F5) was added to the corresponding wells and incubated at 37 °C for 48 h. After 48 h, cells were washed once with PBS and 0.01% PBST before being fixed using 4% PFA (Alfa Aesar, Kandel, Germany) for 15 min at RT. Then, 3% BSA in PBS was added and removed, followed by 2X PBS washes. Permeabilization was performed using 0.5% Triton^®-X100 (Merck Millipore, Darmstadt, Germany) in PBS for 20 min at RT, after which cells were washed with 3% BSA and PBS as mentioned before. Nucleus staining was performed using DAPI solution (0.2 µg/mL, Biolegend) in PBS for 10 min at RT in the dark, followed by 2X PBS washes. Coverslips were mounted carefully using a few drops of Fluoromount (Southern Biotech, Birmingham, AB, USA) on microscopic glass slides. Images were acquired using a confocal microscope (Leica TCS SP8, Wetzlar, Germany), and the mean GFP fluorescence intensity was measured using ImageJ 1.52a (NIH, Bethesda, MD, USA).

Cells were washed once with FACS buffer (PBS−/−, 1% BSA, 2 mM EDTA, 0.1% NaN₃) and after FcR blocking (Human TruStain FcX, Biolegend) co-stained for: HLA-DR-FITC (1:50, clone: L243), CD86-PE-Cy7 (1:20, clone: IT2.2), CD163-APC (1:20, clone: GHI/61); all from eBiosciences (Hertfordshire, UK) and CD206-PE (1:20, clone: 15-2), (Biolegend, London, UK). After 30 min incubation at 4°C in the dark, cells were washed twice with FACS buffer (1500 rpm, 4°C, 5 min) and re-suspended in 500 μl DAPI solution (5ug/ml, Biolegend) for viability and immediately acquired on a BD FACS CANTO II flow cytometer and analyzed using FlowJo 7.6.5 software (Tree Star). Purity of isolated CD14+ cells was checked by staining for CD14-PerCP-Cy5.5 (1:50, clone: HCD14, Biolegend) and was routinely > 95% across all the experiments.

SiHa cells (7 × 10³ cells) were grown overnight at 37 °C with 5% CO₂ in the 8-well slide chamber (Lab-Tek, USA) and transfected with 50 nM siRE1.3–6 using Lipofectamine RNAiMax reagent. After 72 h of transfection, the cells were washed and fixed in cold acetone, and then blocked for an hour with 1% BSA in 1X PBS at room temperature. The air-dry slide was then stained with 1:100 rabbit anti-FOXO3a (phospho S253) antibody and 1:20 rhodamine phalloidin (Invitrogen, USA) mixture, at 4 °C overnight in a moisture chamber. After that, the slide was incubated with a 1:1000 goat polyclonal antibody to rabbit IgG conjugated with FITC (#ab6717; Abcam, USA) for another hour at 37 °C. Then, the slide was stained with 300 nM DAPI solution (#422801; Biolegend, USA) for 5 min at room temperature. Then, the slide was treated with anti-fade fluorescence mounting medium before being observed under the 63 × magnification of the LSM800 airy scan confocal microscope (ZEISS, Germany). The FOXO-positive cells were counted for five fields in each condition to observe the localization. Prism8.0.1 software was used to calculate the Pearson correlation coefficient (r) from the total number of cells and the cell containing nuclear FOXO3a. Three independent experiments were conducted.