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. 1998 Oct;9(10):2891–2904. doi: 10.1091/mbc.9.10.2891

Figure 5.

Figure 5

RacC overexpression stimulates phagocytosis. For A–E, cells were incubated with 1-μm fluorescent latex beads for the indicated time after a 20-min pretreatment with the appropriate drug or DMSO (the diluent). Cells were collected by centrifugation, washed, and prepared for fluorimetry. Shown are the rates obtained by averaging the indicated time points from independent experiments that were performed. The data are reported as fluorescence normalized to total protein/sample to account for potential differences in cell size between the two strains. (A) Untreated cells. RacC overexpression stimulates the rate of 1-μm latex bead uptake three- to fourfold over control Ax2. (B) Cells treated with the PI 3-kinase inhibitor LY294002 (20 μM). Inhibition of PI 3-kinase only partially represses the RacC-induced stimulation of phagocytosis. Control cells treated with PI 3-kinase inhibitors actually display a slight stimulation of bead uptake. (C) Cells treated with calphostin C (0.75 μM), an inhibitor of enzymes containing DAG-binding motifs. Calphostin C treatment completely prevented the uptake of latex beads in control and RacC WT(+) cells. (D and E) Cells treated with bis-indolylmaleimide I (10 μM; D) or chelerythrine (10 μM; E), specific inhibitors of PKC. Phagocytosis was unaffected by treating cells with drugs that specifically inhibit PKC. (F) Cells were incubated with FITC-labeled E. coli, and internalization was measured in the same manner as for latex beads. All values are reported as fluorescence per micrograms of protein ± SEM. Statistical analysis was performed on the data obtained for the 15-min (shown below) and 30-min time points to test the significance of the reported increases and decreases using the Student’s two-tailed t test (Instat, IBM, Armonk, NY). (A) No treatment. Ax2, 14.2 ± 1.1 (n = 12); RacC WT(+), 48.2 ± 3.5 (n = 12). (B) LY294002. Untreated: Ax2, 11.6 ± 1.3 (n = 4); RacC WT(+), 36.7 ± 5.4 (n = 4). LY294002-treated: Ax2, 17.5 ± 2.9 (n = 4); RacC WT(+), 30.7 ± 3.0 (n = 4). (C) Calphostin C. Untreated: Ax2, 16.2 ± 0.8 (n = 3); RacC WT(+), 48.4 ± 7.2 (n = 3). Calphostin C-treated: Ax2, 6.0 ± 1.8 (n = 3); RacC WT(+), 8.9 ± 4.0 (n = 3). (D) Bis-indolylmaleimide. Untreated: Ax2, 16.5 ± 1.0 (n = 3); RacC WT(+), 59.9 ± 5.0 (n = 3). Bis-indolylmaleimide I-treated: Ax2, 17.5 ± 1.4; RacC WT(+), 56.1 ± 2.0 (n = 3). (E) Chelerythrine. Untreated: Ax2, 14.2 ± 2.0 (n = 3); RacC WT(+), 57.6 ± 6.4 (n = 3). Chererythine-treated: Ax2, 18.7 ± 3.7; RacC WT(+), 53.4 ± 5.5 (n = 3). (F) FITC–E. coli: Ax2, 16.4 ± 0.5 (n = 3); RacC WT(+), 38.7 ± 2.4 (n = 3).