TcdB and TcdB autoprocessing mutants cause cell rounding with concentration dependent kinetics.

<p>HeLa cells were treated with multiple concentrations of wild-type and mutant TcdB proteins and imaged every 10 minutes over a 2 hour time course. The percentage of round cells was quantified over six fields for each concentration and time point. The kinetics of rounding induced by TcdB and the TcdB autoprocessing mutants is shown at concentrations of <i>A</i>, 10 pM. <i>B</i>, 100 fM and <i>C</i>, 1 fM. <i>D</i>, Representative images of cells treated with 1 fM TcdB and TcdB autoprocessing mutants for 50 minutes. Green cells are alive; red cells are dead. Images were collected with an Opera High-Throughput Confocal Screening Microscope in an environment-controlled chamber at 37°C, 5% CO₂. Round cells were defined as having an area less than 500 um² and a width-to-length ratio of less than 0.4. Analysis was performed using Columbus Analysis software.