Fluoview fv10i system

For immunocytochemistry, cells on coverslips were fixed in 4% paraformaldehyde-phosphate buffer for 10 min at 48 h after transfection, and then washed three times briefly in PBS. To study intracellular protein localization, cells were permeabilized for 20 min at room temperature with 0.1% Triton X-100 (SIGMA, Kanagawa, Japan) in PBS, blocked with 3% Goat serum (Gibco, Thermo Fisher, MA, USA) in PBS for 20 min, and incubated for 1 h at room temperature with a 1:200 dilution of AntiHalo-Tag pAb (Promega, Tokyo, Japan). Cells were washed three times with PBS, and then incubated for 1 h at room temperature with a 1:200 dilution of the secondary antibody, Alexa Fluor 546 goat anti-rabbit antibody. Total actin was stained with Alexa Fluor 488 phalloidin (Invitrogen, Thermo Fisher, MA, USA), and a 1/1000 dilution of DAPI (KPL, MA, USA) to visualize the nucleus. Cells were washed in PBS 3 times, and coverslips were mounted onto a slide glass with Pro-long Gold Antifade Mountant (Invitrogen, Thermo Fisher, MA, USA). Images were taken with an Olympus Fluoview FV-10i system (Olympus, Okaya, Japan).

HUVECs stably expressing S1PR1-GFP were infected for 48 h with lentivirus to induce shRNA-mediated suppression of the target genes, then transferred onto 35-mm glass-bottomed dishes and incubated for another 24 h. The cells were treated as indicated in the figure legends and fixed in methanol. Nuclei were stained with DAPI (PerkinElmer, #CP81). Confocal fluorescence microscopy was performed using a FluoView FV10i system (Olympus). Intracellular S1PR1 dot numbers were quantified by Matlab software (version R2022a, MathWorks Software).