Ix83 inverted platform

THP1 cells (monocytes) (10 000 cells/well) were seeded in chamber slides (Nunc™ Lab‐Tek™ II Chamber Slide™ System) in RPMI‐1640, supplemented with 10% FBS and 1% penicillin/streptomycin in the presence of PMA (100 ng/mL) for 12 hours, with the aim of differentiating monocytes to macrophages. Thereafter, cells were treated with Exo^Normoxic or Exo^Hypoxic (10 μg each) for 48 hours The effect of exosomes treatment on the THP1 macrophages polarization state was then assessed by arginase‐1 expression (a M2 phenotype marker protein) through confocal microscopy as described earlier.31 In brief, cells were incubated with primary antibody (anti‐arginase) followed by secondary antibody conjugated with Alexa Fluor, followed by staining with Prolong^® Gold Antifade Reagent with DAPI. Fluorescent images were captured using an Olympus FV1200 SPECTRAL Laser scanning Confocal Microscope at 60× objective lens (Olympus IX83 inverted platform).

PC3 or HEK293 cells were transiently transfected with YFP-SAMHD1 mutants as described above and allowed to rest for 24 h. Cells (∼5 × 10⁴ cells per well) were plated in 4-well chamber slides with removable wells and allowed to grow for 48 h before stimulation with 1 µM LPA (Nunc™ Lab-Tek™ II Chamber Slide™ System). Cells were then fixed for 15 min with 10% formalin, washed 3 times with wash buffer (2% FBS, 0.1 M glycine, PBS-T), permeabilized for 15 min with 0.1% Triton-X100 in PBS-T, and washed 3 times with PBS-T. After removing the chamber wells, cells were mounted under coverslips with ProLong® Gold Antifade Reagent with DAPI. Images were collected with an Olympus FV1200 Spectral Laser Scanning Confocal Microscope with an Olympus IX83 inverted platform. Identifying labels were removed from the images and given to two independent researchers to count the number of fluorescent cells and the number of fluorescent cells with cytoplasmic SAMHD1 per frame. A minimum of 200 cells per time point were counted from 3 (HEK293) or 4 (PC3) independent experiments. Statistical analysis was performed by Fisher’s exact test using RStudio (R Version 4.0.2, RStudio Version 1.1.453) (RStudio Team, 2020 ).