Macropinocytosis assay

NIH3T3 cells were seeded as described above and starved overnight in DMEM supplemented with 0.2% BSA and 0.5% FBS. PDGF-BB (0.5 nM) and 70 kDa TMR–dextran (1 mg/ml) were added to the cells for 30 min at 37°C. MDA-MB-231 cells were seeded on glass and cultured overnight in DMEM supplemented with 0.2% BSA, and then incubated with HGF (50 ng/ml) and 70 kDa TMR–dextran (1 mg/ml) for 30 min at 37°C. BT-549 and PC3 cells were plated on glass and grown in complete medium overnight. Cells were incubated with 70 kDa TMR-dextran (1 mg/ml) for 30 min at 37°C. In all cases, dishes were transferred to ice, washed five times with ice-cold PBS, fixed using 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS and mounted. Image acquisition was performed as described above, using a 40× (0.75 NA) objective; for PC3 cells, 0.77 µm sections were taken with a 63×1.4 N.A. objective on a Leica SP5 AOBS confocal microscope. Images were analyzed using ImageJ software as described above, except that macropinosomes were defined as particles greater than 0.75 μm in diameter using the Analyze Particle function.