Fig. 6. Cholesterol is required for the TPA-induced ruffling. (A,B) Serum-starved A431 cells were incubated with 5 mM mßCD for 30 minutes prior to addition of 0.5 µM TPA. After 4 or 10 minutes at 37°C the cells were fixed, permeabilized and stained with rhodamine-labeled phalloidin to visualize filamentous actin. In C and D, serum-starved A431 cells were incubated with a 1:1 mixture of mßCD and mßCD/chol (which does not change the cellular cholesterol content) and mßCD/chol, respectively, for 30 minutes prior to addition of 0.1 µM TPA. After 10 minutes at 37°C the cells were fixed, permeabilized and stained with rhodamine-labeled phalloidin to visualize filamentous actin.