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Fig. 5. Overexpression of a catalytically inactive PLD mutant affects stress fibre formation. (A) C2C12 cells were transfected with HA-tagged catalytically inactive DN-PLD or empty vector. Following serum starvation, the cells were stimulated with S1P in the presence of ethanol and processed as described in the Materials and Methods. PLD activity is reported as percentage of PtdEtOH on PtdCho. Data are mean±s.e.m. of three independent experiments (*P<0.05). Inset: western blot shows the expression of recombinant protein. Blot representative of three experiments with similar results. Time-lapse analysis of myoblasts expressing GFP-actin transfected with DN-PLD by two-photon excitation fluorescence microscopy (B,C). Images were taken 1 minute (B) and 30 minutes after (C) the application of 1 µM S1P. A clear reduction of stress fibres is evident in cells with reduced PLD activity.





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