Fig. 9

Western blot analysis to determine the apoptotic mechanism of doxorubicin a DOX causes cytochrome c release from mitochondria. Y-79 cells were treated with 5 μM of native DOX and equivalent concentrations of DOX in NPs (DOX-CNP and DOX-CNP-FA) for 2, 24, and 48 h. Cytosolic and mitochondrial fractions from intact cells were prepared and analyzed for cytochrome c levels by immunoblotting using an anti-cytochrome c antibody. Samples obtained from the time course of different fractions (cytosol or mitochondria) were adjusted for equal protein loading by determination of concentrations with the Bradford assay. β-actin was used as a standard for the equal loading of protein in the lanes. b DOX activates caspase-9 and caspase-3. Y-79 cultures were treated with 5 μM of native DOX and equivalent concentrations of DOX in NPs (DOX-CNP and DOX-CNP-FA) for 48 h. Thereafter, the cells were harvested and lysed at the indicated times after treatment, and the protein extracts were extracted and analyzed by western blot for the presence of activated caspase-9 and caspase-3. β-actin was used as a standard for the equal loading of protein in the lanes