S1P activates myosin to promote bleb-based motility. (a and b) WT naive CD4⁺ T cells (previously cultured overnight in media with 10% CS-FBS) were stimulated with S1P (100 nM) or CCL19 (100 ng/ml) for the indicated times. Cell lysates were immunoblotted for phospho-MLC (ppMLC; Thr18+Ser19), GAPDH, phospho-AKT (pAKT; Ser473), and total AKT. Band intensities were quantified, and the ratio of ppMLC to GAPDH signal was determined and normalized to 1 for the unstimulated control (indicated with a dotted line). Pooled data from four independent experiments are shown as means ± SD (b). For statistical analysis, variation of each experimental sample from the starting value of 1 was tested using a one-sample t test. (c–e) WT naive CD4⁺ T cells (previously cultured overnight in media with 10% CS-FBS) were imaged by DIC microscopy on VCAM-coated chambers before and after stimulation with S1P (100 nM). Where indicated, cells were pretreated with BBS (5 µM) for 10 min. Scale bars, 5 μm. Time in min:s and relative to point of stimulation. Full arrows indicate direction of migration, arrowheads indicate lamellipodia, asterisks indicate blebs, and # indicates random pseudopodial protrusions. (f and g) Blebs were manually counted, and displacement was tracked from DIC movies. Where indicated, cells were pretreated with BBS (5 µM) for 10 min. Each dot corresponds to a cell pooled from two independent experiments. Means (horizontal bars) of groups compared using an ordinary one-way ANOVA with a Tukey correction for multiple comparisons. UT, untreated. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.