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Fig. 2. Phagocytosis and endocytosis are affected by down regulation of EhCaBP1. (A) Fluid-phase endocytosis in EhCaBP1-AS and EhCaBP1-S cells. Transformed cells were grown in 20 µg/ml hygromycin with or without 5 µg/ml tetracycline for 48 hours. For the FITC-dextran uptake assay, 2x10⁵ cells were incubated with 1 mg/ml FITC-dextran in PBS for 30 minutes followed by thorough washing. Quantitative analysis of the fluorescent images was carried out by counting the number of vesicles containing fluorescent FITC-dextran in each cell. The data represent percentage mean (±s.d.) of fluorescent-labeled vesicles per cell. Ten cells were randomly selected per slide and counting of engulfed beads was done for five separate slides for each cell type. (B) Erythrophagocytosis in EhCaBP1-AS and EhCaBP1-S cells. Transformed cells were grown in 20 µg/ml hygromycin with or without 5 µg/ml tetracycline for 48 hours. The cells (2x10⁶) were incubated with red blood cells (RBCs: 2x10⁸) for the indicated times followed by washing with water to remove adhering cells. The amoebae with engulfed RBCs were then lysed with formic acid. The amount of heme was determined by measuring the optical density at 400 nm for the different cell types indicated. Values are mean ± s.d. (C) The number of RBCs internalized for each amoeba cell-type. The data from sector B was used to compute the number of RBCs internalized as described in Materials and Methods.

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