Fig. 4. Cholesterol is required for the TPA-induced macropinocytosis. (A,B). A431 cells were serum-starved for 4 hours, preincubated for 30 minutes at 37°C with 5 mM mßCD to remove cholesterol and then for 15 minutes at 37°C with HRP (10 mg/ml) in the presence of both 5 mM mßCD and 1 µM TPA (CDx-TPA). It is evident how treatment with mßCD reduces the expression of membrane ruffles at the free surface (FS in B) and prevents the TPA-induced macropinocytosis of HRP from this surface as well as formation of a more complex membrane structure at the lateral membranes facing the intercellular spaces (IS). Moreover, only few caveolea are present. In C and D, serumstarved A431 cells were preincubated for 30 minutes at 37°C with a 1:1 mixture of 2.5 mM mßCD and 2.5 mM mßCD/chol (a mixture that does not change the cellular cholesterol content) and then for 15 minutes at 37°C with HRP in the presence of both TPA and the mßCD mixture (CDx-CDxCh-TPA). Under this condition closure of ruffles (arrows) at the free surface (FS) and the formation of HRP-containing macropinosomes (Mp) take place, complex membrane structures are formed at the lateral membranes at the intercellular spaces (IS), and caveolea are also present at these membranes (arrowheads in D). Bars, 1 µm.