Fig. 4: Sphingosine kinases and S1PR3 are required to maintain membrane homeostasis in human/mouse cells.

A Average GP index from several images of NT, Sphk1 and Sphk2 siRNA-treated HEK293 cells challenged with PA 200 µM and stained with the Laurdan dye. n = 20 separate images analyzed for each condition. B, C Pseudocolor images and average GP index from several images of NT and Sphk1 + 2 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM or ± OA 200 µM. In C, n = 20 separate images analyzed for each condition. D, E Schematic representation of S1P signaling and S1PR pharmacological modulators used in this work. F Relative expression of S1PRs in WT MEFs measured by qPCR. n = 3 technical replicates per condition. G, H Pseudocolor images and average GP index from several images of WT MEFs treated with vehicle, PA 400 µM, PA 200 µM ± JTE-013 5 µM (S1PR2 antagonist), ± TY52156 5 µM (S1PR3 antagonist). In H, for each condition from left to right, n = 15, 5, 15, 15 and 15 separate images analyzed. I Average GP index from several images of NT, AdipoR2, S1PR2, S1PR3 siRNA HEK293 cells treated with PA 200 µM ± S1P 1 µM. For each condition from left to right, n = 10, 10, 15, 10, 14 and 15 separate images analyzed. Data are represented as mean ± SEM, except F: ± SD. t-tests (two sided and assuming normality) were used to identify significant differences between treatments. *p < 0.05, **p < 0.01, ***p < 0.001. See also Supplementary Fig. 5 and Supplementary Data 5. Source data are provided as a Source Data file.