Annexin V/PI-staining of TTC-reactive REH cells and control cells treated with TTC fusion proteins and equivalent control proteins.

<p>The assay was carried out after incubating the TTC-reactive REH cells (A) or control REH cells (B) with 50 nM of each protein for 72 h at 37°C. The induction of apoptosis was measured by FACS after annexin V/PI staining. Representative dot blots are shown with the cells distributed as follows: lower left = viable cells, lower right = early apoptotic cells, upper right = late apoptotic cells. (C)The Induction of apoptosis following treatment with TTC-ETA' and GrB(R201K)-TTC. TTC-reactive REH cells (left) and the control REH cells (right) were incubated with 50 nM TTC-ETA', Mock-ETA', GrB(R201K)-TTC or GrB(R201K)-Mock, with a buffer-only negative control and a camptothecin positive control for 72 h at 37°C. The sum of early and late apoptotic cells ( ± SD) from three independent experiments carried out in duplicate is presented for each protein. The statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test (***: p ≤ 0.001, **: p ≤ 0.01).