SNX5 levels influence macropinocytosis activity. (A) HEK-FlpIn parental (white bars) and HEK-GFP-SNX5 (grey bars) cells were serum-starved for 16 h and pulsed with 100 μg/ml dextran (10,000 Da) conjugated to tetramethylrhodamine for 5 min in the presence or absence of 100 ng/ml EGF at 37°C prior to fixation in 4% PFA. Macropinosomes were identified as dextran-positive structures > 0.5 μm in diameter, and counted using an automated image analysis protocol as described in Methods. The mean number of macropinosomes per 100 cells was determined over 500 cell triplicates for each condition (B) HEK-GFP-SNX5 cells were serum-starved overnight and either left untreated or treated with 100 ng/ml EGF in the presence and absence of 100 nM of AG1478, or in the presence of 0.001% DMSO (carrier control), as indicated. Cells were fixed and permeabilised and stained with anti-human EEA1 and Alexa568-conjugated goat anti-mouse IgG. Macropinosomes were identified as large >1 μm EEA1 positive-structures, as described in text. The mean number of macropinosomes from a triplicate of 100 cells was determined without knowledge of the identity of sample. Each experiment was repeated twice. Shown is mean and error bars represent standard deviation. ** p < 0.05, *** p < 0.005.