Fig. 3.

Combined treatment with KCC-07 and DNA damaging reagents further induces cell death. (A) Changes in cell cycle phases following KCC-07 and/or DNA damaging reagent treatment. The left bar graphs represent percentages of each cell cycle phase, and the right graphs represent cell cycle profiles analyzed by FACS. G2/M arrest is the main response to DNA damaging reagent treatments, and combined treatments with KCC-07 and DNA damaging reagents shift the cell cycle profile toward G1 arrest except for the U-87MG cell line with phleomycin (Phleo) treatment, to which it is less sensitive. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (B) Changes in sub-G1 phase following KCC-07 and/or DNA damaging reagent treatment (from Fig. 3A above). Cell populations in the sub-G1 phase, which indicate cell death, include both floating cells in the culture media and cells attached to culture plates. Combined treatments with KCC-07 and DNA damaging reagents show additive effects on neural tumor cell death in general. Representative data are shown in the graphs (n=3). Each experiment was repeated twice independently. (C) Real-time cell death analysis by IncuCyte. The left line graphs indicate red-stained cell population during exposure to KCC-07 and/or DNA damaging reagents in real-time tracking. Only cells attached to culture plates were measured, due to the technical limitation of this analysis. The right panels show typical cell shape and red staining at the final time points. SH-SY5Y cells aggregate when they are exposed to KCC-07, which U-87MG cells do not. This might reflect higher sensitivity to KCC-07 treatment (see Fig. 1A). Representative data are shown in the graphs (n=3, each sample imaged at nine different spots in real-time). Each experiment was repeated twice independently. The experimental conditions of drug treatments are indicated in the figure. NS, not significant compared to controls.