Fig. 3

Inhibition of S1P generation or function with SK1-II (a and b) and SphK1 knockdown (c and d) attenuates DR5 silencing-induced cell invasion accompanied with blockage of DR5 knockdown-induced activation of JNK and ERK signaling and elevation of TRAF2 and MMP1 (e). a and b, A549 cells transfected with control (Ctrl) or DR5 siRNA were plated after 12 h in Matrigel invasion chambers for cell invasion assays and then exposed to the given concentrations of SK1-II in the bottom wells for an additional 36 h. The invading cells were stained, photographed and measured (a). Moreover A549 cells were seeded in 96-well plates and exposed to different concentrations of SK1-II for about 48 h, and cell numbers were measured with the MTS assay (b). The data are means ± SDs of triplicate determinations. c and d, A549 cells transfected with the indicated siRNAs alone or in combinations were seeded in 12-well plates for Western blotting to detect the given proteins (c) and in Matrigel invasion chambers for cell invasion assays (d) after approximately 48 h incubation. The data are means ± SDs of duplicate determinations from a representative experiment. The experiments were conducted 2-3 times with similar results. e, A549 cells were transfected with control (Ctrl) and DR5 siRNA and after 24 h were exposed to different doses of SK1-II as indicated for an additional 15 h. The cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the indicated proteins