Pseudolaric acid B(PAB),a major biologically active component of "TuJinPi"(the root bark of Pseudolarix kaemferi Gordon),exhibited cytotoxicity in many human tumor cell lines.High content analysis(HCA) is a fluorescence microscopy-based automated technology used for quantitative analysis of multiple targets in cells.HCA could yield rich information about the temporal-spatial dynamics of the fluorescence-labeled cell constituents.The mechanism of inhibitory effects of pseudolaric acid B on human breast cancer MCF-7 cells was explored by high content analysis and flow cytometry.As shown by sulforhodamine B assay,PAB inhibited the proliferation of MCF-7 cells in a dose-dependent and time-dependent manner,and the 50% inhibition concentration(IC50) for 72 h was(1.80±0.33) μmol/L.Flow cytometry(propidium iodide staining) showed that,after treatment with PAB for 24 h,the proportion of MCF-7 cells at G2/M phase could increase to about 93%.Flow cytometry(annexin V-FITC and propidium iodide staining) showed that,PAB induced apoptosis of MCF-7 cells.High content analysis showed that: after treatment with PAB for 16 h,the mitotic index of MCF-7 could increase to about 40%,and cyclin B1 was upregulated;PAB caused dose-dependent disassembly of microtubules and inhibited the formation of mitotic bipolar spindles;PAB induced increase of mitochondrial mass;PAB induced grape-like giant nuclei indicating mitotic slippage in MCF-7 cells.These results suggest that PAB inhibits MCF-7 cell proliferation and induces apoptosis,these inhibitory effects may be related to disassembly of microtubules,spindle abnormalities,mitotic arrest and increase of mitochondrial mass.