Figure 4: MDA-CM obtained from SphK1 knocked-down cells does not inhibit cell invasion.

(a) MDA-MB-231 cells were incubated for 24 h with MDA-CM obtained from PF-543-treated MDA-MB-231 cells and stained for actin. Scale bar, 100 μm. (b) Transwell invasion assays were performed with MDA-CM obtained from PF-543-treated cells. Values represent mean ± s.e.m. of triplicate samples. ***p < 0.001. (c) MDA-MB-231 cells were transfected with a 20 nM of mock or SphK1 siRNA and incubated for 72 h. Extent of decrease of SphK1 mRNA was quantified by real-time PCR. Values represent mean ± s.e.m. ***p < 0.001. (d) Measured S1P concentration of CM obtained from mock or SphK1 siRNA-transfected cells using ELISA. Values represent mean ± s.e.m. of triplicate independent experiments. ***p < 0.001 (e) MDA-MB-231 cells were incubated for 24 h with MDA-CM obtained from mock or SphK1 siRNA-transfected cells as indicated. After incubation, cells were fixed and stained for actin. Scale bar, 50 μm. (f) Transwell invasion assay was performed for 24 h with MDA-CM obtained from indicated condition for 24 h. Values represent mean ± s.e.m. of triplicate independent experiments. *** p < 0.001.