S1PR1 localization. (A and B) HUVECs coexpressing WT-S1PR1-GFP and Y¹⁴³D-S1PR1-GFP or Y¹⁴³F-S1PR1-GFP along with stargazin (membrane marker). At 24 h, cells were fixed and stained for DAPI, and images were acquired using a confocal microscope. A representative image of receptor localization in the cell is shown in A, whereas B shows the quantification of the individual data points representing receptor expression along with mean ± SD (5–8 cells/group). Scale bar, 5 μm. Gray background indicates the area outside the image. (C and D) Time-lapse images of Y¹⁴³D-S1PR1–Dendra2 before (green, rectangles) and after (red, rectangles) photoconversion at time 0 for basal (C) and after the addition of S1P (1 μM). Time is given in minutes. A representative image with time lapse of receptor localization in the cell is shown in C, while D shows the quantification of the individual data points representing receptor expression along with mean ± SD (5–6 cells). Scale bar, 10 μm. Data are from experiments that were repeated at least two times. Significance determined by one-way ANOVA followed by multiple comparisons between groups. A.U, arbitrary units; UD, undetectable.