Fig. 5

Effect of derivative 21 on the activation of the caspase cascade. a HCT116 cells were treated with 50 μM derivative 21 for various durations of time (0–24 h), and whole-cell lysates were subjected to immunoblotting using antibodies specific for the cleaved caspases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to ensure the same amount of protein loading. b HCT116 cells cultured on coverslips were treated with 50 μM derivative 21 for 12 h. The cells were fixed and incubated with antibodies against α/β-tubulin and cleaved caspase-7 for 2 h, followed by incubation with AlexaFluor 488- (green signal) and AlexaFluor 555-conjugated (red signal) secondary antibodies for 30 min. Nuclear DNA was stained with 0.1 μg/mL Hoechst 33,258 for 10 min (blue signal). Arrows indicate the apoptotic nuclear fragments. Fluorescence-positive cells were examined under an EVOS FL^® fluorescence microscope. Dotted boxes are enlarged on the right. Size bars, 50 μm