FIG. 5.

Results of the viability testing of ECP-treated cells. Cells were treated as described in the legend to Fig. 3 and were then incubated for 3 to 48 h before the addition of 33 μl of an MTT solution (5 mg ml⁻¹) from the Vybrant MTT cell proliferation assay kit (Molecular Probes, The Netherlands). The optical densities at 570 nm of triplicate samples were determined. Cell viability is expressed as a mean percentage of the MTT reduction to formazan relative to the activity of untreated cells (defined as 100%) at each time point. (A) Kinetics of the cytotoxicity of LGP32 ECPs to Bge and NIH cells. The upper curve shows that despite the rounding of Bge cells after ECP treatment, the capacity to metabolize MTT was altered only slightly. In contrast, the ECPs induced a continuous decrease of MTT metabolism by NIH cells. (B) Kinetics of the cytotoxicity of mutant ECPs to NIH cells. Cells were incubated with equivalent amounts of ECPs normalized based on the content of total protein and were treated with MTT as described above. The Δvsm strain and the Δvsm-1062 double mutant displayed moderate cytotoxicity, indicating that VSA1062 is not a factor of virulence in the ECPs. Indeed, the ECPs of the Δ1062 strain and LGP32 were equally cytotoxic. Interestingly, the MTT reduction assay provided essentially the same percentages of viability for NIH cells maintained in suspension as for cells treated with virulent ECPs.