Sphingolipids S1P and SPC cause vimentin reorganisation. (A) Representative immunofluorescence images showing that sphingolipid treatment causes cell rounding and reorganisation of the vimentin and actin cytoskeleton in C643 cells. Cells were co-labelled for vimentin (magenta) and actin using fluorescently conjugated phalloidin (green). White squares on the vimentin images indicate the area of the magnified images shown in the inset. Scale bars: 20 μm (main images); 5 µm (inset). (B) 3D projections of confocal stacks of whole-cell-stained (CFSE) C643 cells show rounding effects of S1P and SPC treatment. Cells grown on coverslips were labelled with CFSE and treated with vehicle control (C), S1P (100 nM) or SPC (1 μM) for 30 min. Cells were fixed and CFSE stain was imaged with confocal microscope. 3D surface rendering projections were created with BioImageXD. (C) S1P and SPC decrease the cell perimeter and S1P decreases cell volume. The 3D image stacks described in B were segmented to identify whole cells using BioImageXD. Cell volumes were measured from six stacks per treatment in two separate experiments (minimum 300 cells per treatment). The length of the cell perimeter was measured with CellProfiler. *P<0.05, **P<0.01 (Student's t-test).

Kinetics of S1P- and SPC-induced vimentin S71 phosphorylation. MDA-MB-435S and C643 cells were treated with S1P (100 nM) or SPC (10 µM) for the times indicated and then the phosphorylation of vimentin S71 was analysed by western blotting. Results are mean±s.e.m., n=3. *P<0.05, **P<0.01 between sphingolipid treatment and vehicle control (C) (one-way ANOVA with Dunnett's post-hoc test).

S1P and SPC act through S1P₂ to phosphorylate vimentin and inhibit cell migration. (A) Knockdown of S1P₂ prevents S1P- and SPC-induced vimentin S71 phosphorylation. MDA-MB-435S cells transfected with siControl or siS1P₂ were treated with vehicle control (C), S1P (100 nM, 10 min) or SPC (10 µM, 1 h). Results are mean±s.e.m., n=5. (B) Inhibition of S1P₂ prevents S1P- and SPC-induced vimentin S71 phosphorylation (pS71-Vim). MDA-MB-435S cells were pre-treated with the S1P₂ inhibitor JTE013 (10 µM, 1 h) and then treated with vehicle control (C), S1P (100 nM, 10 min) or SPC (10 µM, 1 h). Results are mean±s.e.m., n=3. (C) Representative immunofluorescence images showing S1P₂ inhibition with JTE013 prevents S1P and SPC induced cell rounding and reorganisation of vimentin in MDA-MB-435S cells. Cells were pre-treated with JTE013 (10 µM, 1 h) as indicated before treatment with vehicle control (CON), S1P (100 nM, 30 min) or SPC (1 µM, 1 h). Cells were co-labelled for vimentin (magenta) and actin using fluorescently conjugated phalloidin (green). Scale bars: 20 μm. (D) Single-cell tracking shows that S1P and SPC inhibits the movement of MDA-MB-435S cells which is rescued by S1P₂ inhibition. Cells were treated with vehicle control (C), S1P (100 nM) or SPC (1 µM) and JTE013 (10 µM) combined (S1P or SPC +JTE; plots for JTE alone not shown). Plots show the tracks of 60 randomly selected cells from three independent experiments. Cells were tracked for 6 h and wide-field images were obtained every 45 min. The intersection of the x- and y-axis is the starting point for each cell. (E) S1P and SPC inhibit velocity of MDA-MB-435S movement and this is rescued by S1P₂ inhibition. Graph shows the velocity of the cells tracked in D. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni's post-hoc test (A,B,E) or Student's t-test (A)).

S1P and SPC act through ROCK to induce vimentin phosphorylation at S71, cause vimentin reorganisation and inhibit cell movement. (A) Inhibition of ROCK prevents S1P- and SPC-induced vimentin S71 phosphorylation (pS71-Vim). MDA-MB-435S cells were pre-treated with ROCK inhibitor Y27632 (10 µM, 1 h) and treated with vehicle control (C), S1P (100 nM, 10 min) or SPC (10 µM, 1 h). Whole-cell lysates were prepared and used for western blotting. Results are mean±s.e.m., n=3. (B) ROCK inhibition with Y27632 prevents the S1P- and SPC-induced cell rounding and perinuclear reorganisation of vimentin in MDA-MB-435S cells. Cells were pre-treated with Y27632 (10 µM, overnight) as indicated before treatment with vehicle control (C), S1P (100 nM, 30 min) or SPC (1 µM, 1 h). Cells were co-labelled for vimentin (magenta) and actin using fluorescently-conjugated phalloidin (green). Scale bars: 20 μm. (C) S1P and SPC inhibit the movement of MDA-MB-435S cells which is rescued by ROCK inhibition. Cells were treated with vehicle control (C), S1P (100 nM) or SPC (1 µM) and Y27632 (10 µM) combined (S1P or SPC+Y2763, plots for Y27632 alone not shown). Plots show the tracks of 60 randomly selected cells from three independent experiments. Cells were tracked for 6 h and wide-field images were obtained every 45 min using Cell-IQ. The intersection of the x- and y-axis is the starting point for each cell. (D) S1P and SPC inhibit velocity of MDA-MB-435S and this is attenuated by ROCK inhibition. Graph shows the velocity of the cells tracked in C). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni's post-hoc test).

Vimentin is important for the inhibitory effects of sphingolipids on cell migration. (A) Sphingolipids induce vimentin S71 phosphorylation (pS71-Vim) in WT MEFs. Cells were treated with S1P (100 nM) or SPC (10 µM) for the times indicated and phosphorylation of vimentin S71 was analysed by western blotting. Images shown are representative of three separate experiments. (B) Sphingolipid treatment inhibits chemotactic migration in WT MEFs but not in vimentin KO MEFs. Cells were allowed to migrate in Boyden chambers towards 10% lipid-stripped serum and vehicle control (C), S1P (10 nM) or SPC (1 μM). Results are mean±s.e.m., n=5. (C) Western blot to show vimentin levels in the vimentin WT and KO MEFs. (D) Expression of the unphosphorylatable S71A vimentin mutant in MDA-MB-435S cells attenuates the inhibitory effect of S1P and SPC on chemotactic cell migration. Cells were double transfected with mCherry–vimentin plasmids and migration experiments were performed as described in Fig. 1A. Results are mean±s.e.m., n=3. (E) Western blot to show the representative protein levels of mCherry–vimentin empty vector (EV), mCherry–vimentin WT and the unphosphorylatable mCherry–vimentin S71A mutant (S71A) compared to the endogenous vimentin levels in MDA-MB-435S cells. (F) Representative immunofluorescence images of the cells expressing the vectors described in E in MDA-MB-435S cells. Co-labelling with fluorescently conjugated phalloidin was used to indicate cell shape. Scale bars: 20 μm. (G) Graphs showing the percentage of transfected cells for each of the vectors. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Bonferroni's post-hoc test).