FIGURE 8.

Differential effects of S1P, Sph, and long-chain ceramides on ERM. A, HeLa cells were treated with bSMase (25 milliunits/ml), bCDase (20 milliunits/ml), or both of them, and pretreated with either vehicle or sphingosine kinase inhibitor (SKI, 10 mm for 6 h), and the levels of Sph and S1P, as well as their dihydro forms were measured. B, phospho-ERM and total ezrin were measured by Western blotting in HeLa cells after being treat with vehicle, bSMase, bSMase, and bCDase together, exogenous Sph (5 μm), exogenous S1P (1 nm), and with bSMase (25 milliunits/ml) and bCDase (20 milliunits/ml) in cells pretreated 6 h with SKI. C, phospho-ERM levels after being treated with S1P (1 nm) or Sph (5 μm) in presence or absence of sphingosine kinase inhibitor (SKI, 10 mm, 6-h pretreatment). D, phospho-ERM levels after being treated with different doses of S1P or Sph. E, HeLa cells were treated in the same way that in B and stained for phospho-ERM (green). Nuclei (stained with Draq5, blue) and F-actin (stained with rhodamine phalloidin, red) also were visualized.