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. 2016 Feb 15;6:21564. doi: 10.1038/srep21564

Figure 5. ECM rigidity-dependent SphK1 expression affects cell invasion and adhesion.

Figure 5

Expression of SphK1 mRNA in MDA-MB-231 cells cultured on PAG with different rigidities as indicated (a) and in 3D collagen matrices with collagen concentration as indicated (b), were verified by real-time PCR. Values represent mean ± s.e.m. of triplicate experiments ***p < 0.001. (c) Measurement of S1P concentration in MDA-CM is carried out using ELISA. Values represent mean ± s.e.m. of triplicate independent experiments. ***p < 0.001 (d) MDA-MB-231 cells were incubated for 24 h with MDA-CM obtained from PAG or 3D collagen matrices, as indicated. After incubation, samples were stained for actin. Scale bar, 50 μm. (e) Transwell invasion assays were performed with MDA-CM used in (c). Values represent mean ± s.e.m. of triplicate independent experiments. ***p < 0.001. (f) MDA-MD-231 cells were plated on 96-well, 50 μg/ml collagen-coated plates and incubated for 60 min with MDA-CM obtained from various conditions as indicated. Non-adherent cells were washed out and adherent cells were fixed and stained with crystal violet. Adherent cells were photographed under a light microscope using a 10X objective lens. (g) Adherent cells were measured at 550 nm using a multi-plate reader. Values represent mean ± s.e.m. of triplicate independent experiments. ***p < 0.001.