FIG. 5.

Early CPE effects on CaCo-2 cells treated with toxin in the presence of caspase or oncosis inhibitors. (A) Specific binding of ¹²⁵I-CPE to CaCo-2 cultures pretreated with a specified caspase or oncosisinhibitor and then treated, in the presence or absence of a 200-fold excess of native CPE, at RT for 15 min with 1 μg of ¹²⁵I-CPE per ml dissolved in HBSS. The same concentration of inhibitor (if any) present during the preincubation step was also present during this binding reaction. Nanograms of ¹²⁵I-CPE specifically bound per 10⁶ CaCo-2 cells were then calculated as described in Materials and Methods. The data shown represent the means from three independent experiments. Error bars indicate the standard deviations. Data with no error bars had a standard deviation too small to depict. (B) Cytotoxic effect of CPE. ⁸⁶Rb-labeled CaCo-2 cultures pretreated with a specified caspase or oncosis inhibitor were then treated for 15 min at 37°C with HBSS containing (as indicated) either 1 or 10 μg of CPE per ml, along with the same caspase or oncosis inhibitor (if any) used for preincubation. The bars represent means from three independent experiments. Error bars indicate the standard deviations; data with no error bars had a standard deviation too small to depict. (C) CPE Western immunoblot analysis of large-complex formation in CaCo-2 cells. Confluent CaCo-2 cells were pretreated with a specified caspase or oncosis inhibitor and then treated for 60 min at 37°C with HBSS that contained or did not contain 1 μg of CPE per ml along with the same caspase or oncosis inhibitor (if any) used for pretreatment. Cell lysates were prepared, electrophoresed, and CPE-Western immunoblotted as described in Materials and Methods. Control, CaCo-2 cells treated only with HBSS (no CPE). The arrows indicate the migrations of the ∼200- and ∼155-kDa CPE complexes.