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Figure 8


Fig. 8. Ceramide impairs the PLD-dependent remodelling of cytoskeleton. (A) L6 cells were transfected with either the empty vector or the PLD1-carrying vector, and stimulated or not by 10-7 M AVP for 15 minutes. F-actin was stained with rhodamine-phalloidin, and the percentage of cells forming stress fibers (SF) was assessed. The means ± s.e.m. of 10 fields are shown. **Indicates a significant difference to the empty vector + AVP value, P<0.001. (B) The cells were pretreated for 3 hours with 10 µM C6-ceramide and/or 100 µM DiC8-PA in 1% BSA medium. They were then stimulated or not with 10-7 M AVP for 15 minutes, and SF formation was assessed as above. Only the cells that formed thick parallel and continuous SFs were considered positive. (C) Fluorescence microscopy of F-actin in the above experiment. AVP stimulation induced the formation of regularly arranged, parallel fibers. When treated by ceramide, the AVP-stimulated cells displayed a fainter actin labeling because of a decrease in the number and thickness of the fibers and the presence of incomplete fibers. An irregular organization of actin, with some radial foci, was also noticed. Conversely, in the presence of PA the cells displayed a dense array of parallel fibers.





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