Fig. 3

Effects of cannflavin A on apoptosis and autophagy. Cells were treated for 24 h for apoptosis or 6h for autophagy with either the methanol vehicle or cannflavin A. A Histogram showing the percentage of annexin V–labeled cells and % cells stained for propidium iodide, indicative of apoptosis. Cells were counted from three random fields of view on a fluorescence microscope. **p < 0.01, n of at least 8. B Western blotting analysis was performed using an anti-caspase 3 antibody, and β-tubulin was included as a loading control. The results indicate activation of caspase 3. Figure is a representative blot of n = 3 experiments. C Histogram showing the percentage of cell viability following 24-h cannflavin A treatment in T24 cells in the presence or absence of autophagy inhibitors (100 nM bafilomycin A or the combination of 10 μg/ml E-64d (aloxistatin) and 10 μg/ml (pepstatin A)). The inhibitors were able to revert the cytotoxic effects of cannflavin A. ***p<0.001, n of at least 3