SphK1 modulates invasion in SCC-25 cells. (A) SCC-25 cells with an invasive phenotype have 2-fold higher mean normalized expression (MNE) of SphK1 mRNA compared to parent cells (left). SCC-25 invaded cells have greater activation of AKT (60 kDa) and ERK (42/44 kDa) compared to parent cells (immunoblot, right). (B) Overexpression of human SphK1 in SCC-25 cells (SphK1) significantly increased SphK1 mRNA levels compared to mock conditions (GFP) as measured with qPCR (P < 0.0001, left). SphK1 overexpressing cells displayed greater invasiveness across transwell Matrigel membrane compared to mock-transfected cells (right, invasion assay). (C) SphK1 transient knockdown significantly reduced the number of cells that invaded and transversed the transwell Matrigel membrane (P < 0.05, left). Image of invaded cells transected with scramble sequence (SCR) or SphK1 siRNA (siSphK1) (right). (D) MMP-2 and −9 mRNA were significantly increased in SphK1 overexpressing cells, (P < 0.001 and P < 0.0001, respectively, qPCR). (E, top) Media cultured by SphK1 overexpressing cells have slightly greater amounts of pro-MMP-2 (72 kDa) unstimulated conditions (gelatin zymography). (E, bottom) SphK1 overexpressing SCC-25 cells have reduced E-cadherin levels (135 kDa) compared to control conditions in basal conditions. Means ± SE are presented, *p < 0.05, ***p < 0.001, ****p < 0.0001.