Fig 3.

⁸⁶Rb release experiments. (A) Cytotoxicity screening of rCPE mutants by an ⁸⁶Rb release assay. Confluent Caco-2 cells in 24-well plates were radiolabeled with 4 μCi/well of ⁸⁶RbCl for 4 h at 37°C and then treated at 37°C with 10 μg/ml of the specified purified 6× His-tagged rCPE species. After 15 min, ⁸⁶Rb released into the medium was collected and quantified with a Cobra Quantum gamma counter. Data from this experiment were converted into the percentage of maximal ⁸⁶Rb release (see Materials and Methods). The bars shown in red correspond to significantly attenuated mutants (P < 0.05). Duplicate samples of each variant were tested in three independent experiments. Error bars depict the standard errors. (B) Dose effects of rCPE species on ⁸⁶Rb release. To further characterize the Cys variants showing reduced ⁸⁶Rb release in panel A, ⁸⁶RbCl-labeled Caco-2 cells were treated at 37°C with increasing amounts of the specified rCPE species. Data shown represent the means of three independent experiments, and the error bars show the standard deviations. (C) Cellular morphological damage caused by rCPE species. To confirm the results from the⁸⁶Rb release experiments, a morphological damage assay was performed as described for Fig. 1. Confluent Caco-2 cell monolayers were treated at 37°C for 60 min with 2.5 μg/ml of each specified rCPE species, including nCPE, rCPE, the C186A variant, the F87C variant, the Q97C variant, and the D48A variant. As a control, some Caco-2 cells were treated with HBSS buffer alone (cHBSS; no added rCPE species).