FIGURE 3.

Effects of S1P on AA(−)-induced autophagy. A, HL-60 cells were incubated in AA(−) medium with 20 μm S1P for 1 h. Autophagosome formation was detected by immunostaining of LC-3. Scale bar, 10 μm. B, cells were incubated in AA(+) medium or AA(−) medium with or without 20 μm S1P for 1 h. LC3 protein was detected by Western blot analysis. C, after treatment with AA(+) or AA(−) together with or without S1P for 1 h, the cells were further incubated with 50 μm MDC for 10 min and immediately analyzed by fluorescence microscopy. The density of MDC was assessed using MacSCOPE. Scale bar, 10 μm. D, cells were incubated with AA(−) medium for the indicated times in the absence or presence of 20 μm S1P, and then the lipids were extracted. Ceramide content was measured using the diacylglycerol kinase method. Values are the mean ± S.D. from three independent experiments. *, p < 0.005; ns, no significance.