Figure 5.

Effects of brefeldin A, bafilomycin A1, and monensin on CNF1-induced multinucleation in HEp-2 cells. Cells cultivated in 24-well plates were washed briefly with a HEPES-buffered medium and incubated with brefeldin A (5 μg/ml) for 1 h. This treatment resulted in the total disruption of the Golgi apparatus, as indicated by immunofluorescence labeling with the anti-Golgi mAb CTR433 (see MATERIALS AND METHODS). Cells were then incubated with CNF1 (10⁻¹⁰ M) for 90 min at 37°C still in the presence of brefeldin A. Then the medium was removed, and cells were washed and incubated with fresh medium containing bafilomycin A1 to block further entry of CNF1. Cells were incubated for an additional 24 h, and multinucleation was recorded. Identical experiments were performed with bafilomycin A1 and monensin. Effects of increased concentrations of NH₄Cl on DT activity are given as controls. Results are the average of duplicate determinations.