Figure 4

Treatment with Cas III-ia induces apoptosis in tumor cells. A) Apoptosis of control and 24 h Cas III-ia-treated glioma C6 cells (5, 10, 15 and 20 μg/ml) was determined by TUNEL assay and visualized by confocal microscope (Green-fluoresein and Blue-DAPI). The figures shown are representative of at least three different experiments for each experimental condition. Original magnification x20 for 5 and 10 μg/ml of Cas III-ia, and x40 for 15 and 20 μg/ml of Cas III-ia. B) Expression of capase-8, Bid and Bax proteins from controls and 24 h Cas III-ia–treated C6 glioma cells determined by Western blot. C) Mitochondrial membrane potential from controls and 24 h Cas III-ia–treated C6 glioma cells was measured by accumulated rodamine 123 (R123) fluorescence determined by flow cytometry. Data represent the mean ± SD (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001) of three independent experiments. D) cytosolic cyt c and activation of caspase 3 were determined by Western blot. The figure shown is representative of at least three different experiments for each experimental condition. E) Effect of ZVAD on Cas III-ia induced cytotoxicity in C6 glioma cells. It was determined by MTT assay in control cells and cells treated with Cas III-ia and Cas III-ia + 50μΜ ZVAD for 24 h; data represent the mean ± SD (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001) of three independent experiments.